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Abstract:

The present invention is directed to compounds of the formula:
##STR00001##
or salts thereof or N-oxides and their use in peptide synthesis.

Claims:

1. A compound or salt consisting of an anion and cation, wherein the
compound or the cation of the salt is of the formula ##STR00079##
whereinR1 and R2 taken together with the carbon atoms to which
they are attached form a pyridyl ring, said pyridyl ring may be
unsubstituted or substituted with a lower alkyl group or electron
donating group;Y1 is N;Q1 is N;R14 is a positively charged
electron withdrawing group, ##STR00080## SO2R17, lower alkyl
carbonyl, lower arylalkyl carbonyl, aryl carbonyl, lower alkyl aryl, or
BLK1-AA1 R17 is aryl, aryl lower alkyl or lower
alkyl;AA1 is an amino acid or peptide less a hydrogen atom on the
N-terminus and an OH on the C-terminus;BLK1 is an amino protecting
group,R10 is OR12, lower alkyl, aryl, aryl lower alkyl, lower
cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocyclic
lower alkyl, lower cycloalkenyl, or lower cycloalkenyl lower
alkyl;R11 is OR13, lower alkyl, aryl, aryl lower alkyl, lower
cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocyclic
lower alkyl, lower cycloalkenyl or lower cycloalkenyl lower alkyl;and
R10 and R11 may optionally be connected by a bridging group
selected from the group consisting of O, S, NR30, or
(CHR30)m, wherein each R30 is independently lower alkyl or
hydrogen and m is 1-3; andR12 and R13 are independently lower
alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic,
heterocyclic lower alkyl, lower cycloalkenyl or lower cyclalkenyl lower
alkyl;ring A1 and ring B are independently an aromatic ring
containing 6 to 14 ring carbon atoms or cycloalkenyl or cycloalkyl, each
containing 5 to 14 ring carbon atoms, andRb1, Rc1, Rb2,
Rc2 are independently hydrogen, lower alkyl or electron donating
group;T is CHR31, O, S or NR30; andR31 is hydrogen or
lower alkyl.

2. The salt according to claim 1 wherein R14 is a positively charged
electron withdrawing group.

3. The salt according to claim 2 wherein R14 is an electron
withdrawing group of the formula ##STR00081## whereinR18, R19,
R20, R21, R22, R23 and R24 are independently
hydrogen, lower alkyl, or lower alkoxy lower alkyl or R18 and
R19 taken together with the atoms to which they are attached form a
ring containing up to 6 ring atoms and up to a total of 5 carbon ring
atoms or R20 and R21 taken together with the nitrogen atom to
which they are attached form a 5 or 6 membered nitrogen containing
heterocyclic ring containing up to a total of 5 carbon ring atoms or
R18 and R20 taken together with the nitrogen atom and the
carbon atom to which they are attached form a heterocyclic ring, or
R22 and R23 taken together with the atoms to which they are
attached form a ring containing up to 6 ring atoms and up to a total of 5
carbon atoms or R24 and R25 taken together with the carbon
atoms to which they are attached form a ring containing up to 6 ring
atoms and up to a total of 5 carbon atoms.

4. The salt according to claim 3 wherein R14 is ##STR00082## or
P(NR24R25)3 wherein R19 and R21, R24 and
R25 are independently hydrogen, or lower alkyl or lower alkoxy lower
alkyl; and n1 is 0 or 1.

5. The salt according to claim 4 wherein R19 and R21 or R24
and R25 are the same.

10. The compound according to claim 9 wherein R10 is OR12, lower
alkyl, aryl, or aryl lower alkyl; R11 is OR13, lower alkyl,
aryl; or aryl lower alkyl and R10 and R11 may optionally be
connected by a bridging group selected form the group consisting of O, S,
NH, and (CH2)m; m is 1-3; andR12 and R13 are
independently lower alkyl, aryl, or aryl lower alkyl.

11. The compound according to claim 1 wherein ##STR00086## wherein
R10 and R11 are independently lower alkyl or aryl.

12. The compound according to claim 1 wherein ##STR00087## wherein
R11 and R12 are independently lower alkyl or aryl.

13. The compound or salt according to claim 1 wherein the compound or the
cation of the salt has the formula ##STR00088## wherein R1, R2,
Q1 and R14 are as defined in claim 1.

14. The compound according to claim 1 wherein the compound or the cation
of the salt has the formula ##STR00089## whereinA is N or CR24;D is
CR25 or N;E is CR26 or N;G is CR27 or N;R24,
R25, R26 and R27 are independently hydrogen, a lower alkyl
group or an electron donating group;wherein one of A, D, E and G, is
N;Y1 is N;Q1 is N;R14 is a positively charged electron
withdrawing group, ##STR00090## SO2R17, lower alkyl carbonyl,
aryl carbonyl, loweralkyl aryl, or BLK1-AA1 R17 is aryl,
aryl lower alkyl or lower alkyl;AA1 is an amino acid or peptide less
a hydrogen atom on the N-terminus and an OH on the C-terminus;BLK1
is an amino protecting group,R10 is OR12, lower alkyl, aryl,
aryl lower alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl,
heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl, or lower
cycloalkenyl lower alkyl;R11 is OR13, lower alkyl, aryl, aryl
lower alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl,
heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl or lower
cycloalkenyl lower alkyl;and R10 and R11 may optionally be
connected by a bridging group selected from the group consisting of O, S,
NR30, or (CHR30)m, wherein each R30 is independently
lower alkyl or hydrogen and m is 1-3; andR12 and R13 are
independently lower alkyl, lower cycloalkyl, lower cycloalkyl lower
alkyl, heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl or
lower cyclalkenyl lower alkyl;ring A1 and ring B are independently
an aromatic ring containing 6 to 14 ring carbon atoms or cycloalkenyl or
cycloalkyl, each containing 5 to 14 ring carbon atoms, andRb1,
Rc1, Rb2, Rc2 are independently hydrogen, lower alkyl or
electron donating group;T is (CHR31), O, S or NR31; andR31
is hydrogen or lower alkyl.

15. The compound or salt according to claim 14 where the compound or the
cation has the formula ##STR00091## whereinA is N or CR24;D is
CR25 or N;E is CR26 or N;G is CR27 or N;R24,
R25, R26 and R27 are independently hydrogen or lower
alkyl;wherein one of A, D, E and G, is N;Y1 is N;Q1 is
N;R14 is a positively charged electron withdrawing group,
##STR00092## SO2R17, lower alkyl carbonyl, aryl carbonyl,
loweralkyl aryl, or BLK1-AA1 R17 is aryl, aryl lower alkyl
or lower alkyl;AA1 is an amino acid or peptide less a hydrogen atom
on the N-terminus and an OH on the C-terminus;BLK1 is an amino
protecting group,R10 is OR12, lower alkyl, aryl, aryl lower
alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic,
heterocyclic lower alkyl, lower cycloalkenyl, or lower cycloalkenyl lower
alkyl;R11 is OR13, lower alkyl, aryl, aryl lower alkyl, lower
cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocyclic
lower alkyl, lower cycloalkenyl or lower cycloalkenyl lower alkyl;and
R10 and R11 may optionally be connected by a bridging group
selected from the group consisting of O, S, NR30, or
(CHR30)m, wherein each R30 is independently lower alkyl or
hydrogen and m is 1-3; andR12 and R13 are independently lower
alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic,
heterocyclic lower alkyl, lower cycloalkenyl or lower cyclalkenyl lower
alkyl;ring A1 and ring B are independently an aromatic ring
containing 6 to 14 ring carbon atoms or cycloalkenyl or cycloalkyl, each
containing 5 to 14 ring carbon atoms, andRb1, Rc1, Rb2,
Rc2 are independently hydrogen, lower alkyl or electron donating
group;T is (CHR31), O, S or NR31; andR31 is hydrogen or
lower alkyl.

[0004]The present invention relates to a new process for effecting the
acylation step in amide formation, especially in peptide synthesis.

[0005]2. Description of the Prior Art

[0006]Polypeptides, especially proteins, play a critical role in
fundamental biochemical processes in living cells. Biochemical reactions,
including metabolic reactions, are catalyzed by enzymes, which are
comprised of proteins. These proteins are chiral molecules, and it is
often the case that of the various stereoisomers that may possibly exist,
one is usually the most efficacious.

[0007]Moreover, polypeptides are useful as medicaments. In recent years,
peptides have been found useful in combating various diseases, including
cancer, diabetes, plant toxins, and the like. Additionally, peptides have
shown specific activity as growth promoters, suppressants, antibiotics,
insecticides, contraceptives, anti-hypertensives, sleep inducers,
anti-depressants, analgesics, and so on.

[0008]The synthesis of proteins has always been a challenge to chemists.
However, chemical synthesis offers advantages not realized by genetic
engineering and other biological approaches such as isolation of natural
proteins. First, it is useful in confirming the structure of a protein.
Moreover, protein synthesis is necessary to synthesize analogs, allowing
scientists to evaluate biological activity and/or pharmacological
efficacy in relation to molecular structure.

[0009]Success in the chemical synthesis of peptides relies, in part, on
the use of the appropriate coupling reagents in combination with the
appropriate protecting groups. Especially in peptide synthesis, formation
of the peptide bond between two amino acids requires activation of the
carboxyl group of one of the amino acids before the reaction can occur.
However, the activation step in conjunction with the coupling reaction
causes a serious problem of loss of configuration at the carboxyl residue
which has been activated. Thus, in designing chemical syntheses of
peptides, the objective is to provide the peptide product in good yield
and maintenance of the configurational integrity of the carboxylic
component, i.e., minimal racemization. Thus, the duality of good yield
and minimal or no racemization is difficult to achieve because the best
methods require the acid to be converted to a derivative bearing a good
leaving group. Thus, under normal coupling conditions, there is a loss of
configuration.

[0010]Moreover, current methods of syntheses also tend to produce side
reactions which decrease yield.

[0011]Currently, syntheses of peptides are in solution by classical or
various repetitive methods. Alternatively, peptides may be prepared on a
solid support (Merrifield method). These are all popular techniques in
synthesizing peptides from the coupling of two or more amino acids, in
synthesizing larger peptides from the coupling of amino acids with
smaller peptides or in the coupling of smaller peptides. Solution methods
have the advantage of being easily monitored, allowing purification of
intermediates, if necessary, at any stage. A major drawback, however, is
the relative slow pace of synthesis, with each step being carried out
manually.

[0012]The major advantage of the Merrifield method is its easy automation
so that unattended, computer-controlled machine synthesis is possible.
Unfortunately, the method suffers from an inherent deficiency due to the
insoluble nature of the support on which the synthesis proceeds. Unless
each acylation step occurs with approximately 100% efficiency; mixtures
will inevitably be built up on the polymer. The longer the chain, the
greater will be the contamination by undesired side reactions. Side
products produced in such reactions remain to contaminate the desired
product when it is removed from the polymeric matrix at the end of the
cycle. These current techniques are not useful in preparing peptides of
greater than 40-50 residues; separation of side products from the desired
product becomes increasingly difficult when larger peptides are
synthesized.

[0013]For very long segments (50 or more amino acids), therefore, current
methods are not satisfactory. Often, mixtures are obtained of such
forbidding complexity that it may be difficult or impossible to isolate
the desired peptide.

[0014]The problems enumerated hereinabove may be eliminated if the proper
derivatives of the underlying amino acids and/or the proper conditions
for the coupling reaction could be found. Protecting groups, such as
t-butyloxy-carbonyl (t-Boc) or N-α-(9-fluorenylmethyl)oxycarbonyl
(Fmoc), have been used to minimize side reactions.

[0015]The most commonly used coupling reagents are carbodiimides such as
dicyclohexylcarbodiimides, diisopropylcarbodiimides,
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimides used with various
additives.

[0016][Additives generally inhibit side reactions and reduce racemization.
Heretofore, the most common peptide coupling additive used during peptide
coupling for peptide synthesis is 1-hydroxybenzotriazole (HOBt). This
reagent has been used either in combination with a carbodiimide or other
coupling agent or built into a stand alone reagent, such as
1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP) or an analogous uronium salt. HOBt is applicable to both stepwise
and segment condensations. However, many cases have been encountered in
which HOBt is ineffective, possibly because of steric effects, or low
basicity of the amino component. Especially problematic are segment
couplings at amino acid units other than glycine or proline, since the
problem of racemization may be severe. The related
N-hydroxybenzotriazinone (HOOBt) may provide better protection against
racemization, but it is rarely used due to competing side reactions
involving ring openings. A drawback in the use of BOP is that it produces
a toxic side product, hexamethylphosphorotriamide.

[0018]Another additive that has been used in peptide synthesis is
3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt). HODhbt has
proved to be generally superior to HOBt. Moreover, its use permits one to
follow the completion of the reaction visually by a color change which
occurs when acylation is complete. However, HODhbt has problems
associated therewith due to inherent side reactions.

[0019]Other derivatives, which include
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate,
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,1-tetra-methyluronium
tetrafluoroborate and
[3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)oxy]tris(pyrrolidino)phosphoni-
um hexafluorophosphate also have applications in peptide coupling.

[0021]U.S. Pat. No. 5,644,029 to Carpino discloses, among other things,
the use of compounds of the following formula in promoting peptide
coupling:

##STR00002##

[0022]or N-oxides thereof or salts thereof wherein [0023]R1 and
R2 taken together with the carbon atoms to which they are attached
form a heteroaryl ring wherein said heteroaryl ring is an oxygen, sulfur
or nitrogen containing heteroaromatic containing from 3 and up to a total
of 13 ring carbon atoms, said heteroaryl may be unsubstituted or
substituted with lower alkyl or an electron-donating group; [0024]Y is O,
NR4, CR4R5; [0025]R5 is independently hydrogen or
lower alkyl; [0026]X is CR6R7 or NR6; [0027]R6 and
R7 are independently hydrogen or lower alkyl; or R6 and R7
taken together form an oxo group or when n=O, R4 and R6 taken
together may form a bond between the nitrogen or carbon atom of Y and the
nitrogen or carbon atom of X; [0028]Q is (CR3R9) or (NR8);
[0029]when n is 1, R4 and R8 taken together may form a bond
between the ring carbon or nitrogen atom of Q and the ring carbon or
nitrogen atom of R8; [0030]n is O or 1; [0031]R3 is hydrogen,
lower alkyl carbonyl, aryl carbonyl, lower aryl alkyl carbonyl,

##STR00003##

[0031]a positively charged electron withdrawing group, SO2R14,
or

##STR00004##

[0032]R14 is lower alkyl, aryl or lower arylalkyl; q is 0-3;
[0033]R8 and R9 are independently hydrogen or lower alkyl or
R7 and R8 taken together with the carbon to which they are
attached form an aryl ring, AA1 is an amino acid and BLK is an amino
protecting group, and m is 0 or 1.

[0034]The present inventor has found other coupling agents, which provide
relatively pure products with little, if any, side products being
co-produced and minimal, if any, racemization. Moreover, the reaction
conditions are very mild and the reagents used are easy to prepare. Thus,
by using the compounds of the present invention as additives, the yield
of the peptide s enhanced and little, if any, racemization occurs.

SUMMARY OF THE INVENTION

[0035]The present invention relates to a compound of Formula I and the use
thereof in the preparation of a peptide bond in peptide synthesis, said
compound having the formula:

##STR00005##

[0036]or N-oxide or N-oxide thereof or salt said compounds of Formula I
wherein [0037]R1 and R2 taken together with the carbon atom to
which they are attached form an aryl or a heteroaryl ring, wherein said
aryl ring is an aromatic ring containing 6-14 ring carbon atoms and
heteroaryl ring is an oxygen, sulfur, or nitrogen containing
heteroaromatic containing at least 1 and up to 4 ring heteroatoms
selected form oxygen, nitrogen and sulfur and containing from 3 and up to
a total of 13 ring carbon atoms, said aryl and heteroaryl may each
independently be unsubstituted or substituted with lower alkyl or
electron donating group or electron withdrawing group; [0038]Q is
CR8R9 or NR8; [0039]Y is O, NR4 or CR4R5,
[0040]X is CR6R7 or NR6; [0041]R5 is hydrogen or
lower alkyl; [0042]R4 is hydrogen or lower alkyl or [0043]R4
and R6 may form a bond between X and Y, when Y is NR4 or
CR4R5 and when Q is not present, or R4 and R8 may
form a bond between Y and Q when Y is NR4 or CR4R5 and Q
is present, or R6 and R8 may form a bond between Q and X when Q
is present; [0044]R8 and R9 are independently hydrogen or lower
alkyl, or when Q is present, R8 taken together with R4 may form
a bond between Q and Y, when Y is NR4 or CR4R5 or when Q
is present, R8 and R6 may form a bond between Q and X;
[0045]R6 and R7 are independently hydrogen or lower alkyl or
R6 and R7 taken together form an oxo; or R6, taken
together with R4, may form a bond between Y and X when Q is not
present and Y is NR4 or CR4R5 or R6 and R8 taken
together may form a bond between Q and X when Q is present; but in no
circumstances is there a double bond between X and Q and Q and Y at the
same time; [0046]n is 0 or 1; [0047]R3 is

[0055]In another embodiment, the present invention is directed to a
compound of formula II or to a salt, in which the cationic portion has
the structure of Formula II and to the use of the compound or salt:

##STR00007##

or N-oxides thereof or salts of said compound of Formula II wherein
[0056]R1 and R2 taken together with the carbon atom to which
they are attached form a heteroaryl ring, wherein said heteroaryl is an
heteroaromatic containing at least 1 and up to 4 ring heteroatoms
selected from O, S and N and containing from 3 and up to a total of 13
ring carbon atoms, said heteroaryl may be unsubstituted or substituted
with lower alkyl or electron donating group or electron withdrawing
group; [0057]Y1 is N or CR15; [0058]R15 is H or lower
alkyl; [0059]Q1 is N or CR16; [0060]R16 is H or lower
alkyl; and [0061]R14 is hydrogen, a positively charged electron
withdrawing group, SO2R17, lower alkyl carbonyl, aryl carbonyl,
lower arylalkyl carbonyl, BLK1AA1,

##STR00008##

[0062]R10 is OR12, lower alkyl, aryl, aryl lower alkyl, lower
cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocyclic
lower alkyl, cycloalkenyl or cycloalkenyl lower alkyl; [0063]R11 is
OR13, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl, lower
cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,
cycloalkenyl or cycloalkenyl lower alkyl; or [0064]R10 and R11
may optionally be connected by a bridging group T1 consisting of O,
NR30 or (CHR30)m, wherein R30 is lower alkyl or H and
m is 1-3; [0065]R12 and R13 are independently lower alkyl,
aryl, aryl lower alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl,
heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl or
cycloalkenyl lower alkyl; [0066]rings A1 and B are independently
aromatic rings containing 6 to 14 ring carbon atoms or cycloalkenyl or
cycloalkyl, each containing 5 to 14 ring carbon atoms; [0067]T is O, S,
NR31 or CHR31; [0068]R31 is H or lower alkyl; and
[0069]Rb1, Rc1, Rb2 and Rc2 are independently
hydrogen, lower alkyl or electron donating group; [0070]R17 is aryl,
loweralkyl or lower arylalkyl, AA1 is an amino acid or peptide less
a hydrogen atom on the N-terminus and an OH group on the C-terminus, and
BLK1 is an amino protecting group. In addition, the present
invention is directed to the use of the compounds of Formula II or, when
R14 is a positively charged electron withdrawing group, its salt in
which the cationic position has the structure of Formula II, in the
preparation of peptide.

[0071]An additional embodiment of the present invention is directed to a
process for preparing a peptide bond from the reaction between an amino
compound and an acylating derivative of a carboxylic acid, said amino
compound being an amino acid or peptide and said carboxylic acid being an
N-terminal amino protected amino acid or an N-terminal amino protected
peptide, which comprises reacting said amino compound and said acylating
derivative of a carboxylic acid in the presence of an effective amount of
a compound of formula I or formula II or to a salt, the cationic portion
of which has the structure of Formula II, under conditions effective to
form a peptide bond.

[0072]In another embodiment, the present invention is directed to a
process for forming an amide from the reaction of an organic amine having
a free amino and an acylating derivative of a carboxylic acid, which
comprises reacting said amine with the acylating derivative of the
carboxylic acid with an effective amount of a compound of Formula I or II
or, when R14 is a positively charged electron withdrawing group, a
salt, in which the cation has the structure of Formula II under amide
forming conditions.

[0073]An additional embodiment of the present invention is directed to the
process for synthesizing peptides comprising (a) reacting a first
Na-amino protected amino acid with a peptide synthesis resin under
conditions effective to covalently link the amino acid to the resin, (b)
cleaving the protecting group from the amino acid to form an amine with a
free amino group, (c) reacting said amine with a second N α-amino
protected amino acid in the presence of a peptide forming effective
amount of a compound of Formula I or II or when R14 us a positively
charged electron withdrawing group, to a salt in which the cation has the
structure of Formula II, said reaction being effected under
peptide-forming conditions, (d) repeating steps (b) and (c) until the
desired peptide is obtained and (e) removing the peptide from the resin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0074]As used herein, the term "salt of Formula II" or any reference to a
salt of Formula II refers to a salt of Formula II which consists of an
anion and cation, the cation portion of which is positively charged. The
positive charge may result from protonation, such as in the presence of
an acid of the compound or Formula II or it may result from R14
being a positively charged electron withdrawing group. If reference is to
be made to a salt of Formula II in which the structure of Formula II
contains a positively charged electron withdrawing group, the term
"cation of Formula II" or its equivalent will be used.

[0075]As described hereinabove, an embodiment of the present invention
relates to compounds of Formula I or Formula II or to salts thereof or
N-oxide thereof or cation of Formula I and their use in peptide coupling.
In other words, a first amino acid or a first peptide, each having a free
amino group is coupled with an acylating derivative of either a second
amino acid or a second peptide in the presence of compounds of Formula I
or II or salts thereof or N-oxides thereof or cation of Formula II under
amide forming conditions to form a peptide bond and thus form a larger
peptide.

[0076]As employed herein, the term "heteroaryl" is a heteroaromatic
containing at least one heteroatom ring atom selected from nitrogen,
sulfur and oxygen and up to a maximum of four ring heteroatoms. The
heteroaryl contains from 5 to 14 ring atoms and up to a total of 13 ring
carbon atoms and a total of 18 carbon atoms. The heteroaryl group may be
monocyclic, bicyclic or tricyclic, although it is preferred that the
heteroaryl is bicyclic and especially monocyclic. Also included in this
expression are the benzoheterocyclics. The heteroaryl group preferably
contains no more than two ring heteroatoms, and most preferably contains
one ring heteroatom. The most preferred ring heteroatoms are oxygen and
nitrogen, with nitrogen being the most preferred.

[0077]If nitrogen is a ring atom, N-oxides can also be formed. The present
invention contemplates the N-oxides of the nitrogen containing
heteroaryls.

[0079]When R1 and R2 taken together with the carbons to which
they are attached form a tricyclic heteroaryl group, then the compounds
of Formula I or II is tetracyclic; if a bicyclic heteroaryl group is
formed from R1 and R2 taken together with the carbons to which
they are attached, then the compounds of Formula I or II are tricyclic.
Finally, if R1 and R2 taken together form a monocyclic
heteroaryl group, then the compounds of Formula I or II are bicyclic. It
is preferred that compounds of Formula I and II are tricyclic, and
especially bicyclic.

[0080]The term "heterocyclic", as used herein, when used alone or in
combination with other groups, refers to a heterocyclic ring containing
at least one heteroatom ring atom selected form nitrogen, sulfur and
oxygen up to a maximum of 4 ring heteroatoms and from 5 to 14 ring atoms
and up to a total of 18 carbon atoms. The heterocyclic group may be
monocyclic, bicyclic or tricyclic. It may be completely saturated or it
may be partially unsaturated, i.e., it may contain one or more double
bonds between ring atoms. It is preferred that the heterocyclic group
contains 0, 1, 2, 3 or 4 double bonds. The term heterocyclic also
includes heteroaryl, as defined herein. Moreover, it is preferred that
the heterocyclic moiety contains no more than two ring heteroatoms and
most preferably no more than one ring heteroatom. Examples include
tetrahydrofuran, morpholinyl, piperazinyl, 2-tetrahydro quinolyl,
3-tetrahydroquinolyl, 6-tetrahydroquinolyl or 7-tetrahydroquinolyl and
the like.

[0081]The term "lower alkyl," when used alone or in combination with other
groups, refers to a carbon chain containing from one to six carbon atoms.
It may be a straight chain or branched and includes such groups as
methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl,
n-pentyl, amyl, hexyl and the like. The preferred lower alkyl group
contains from 1-3 carbon atoms, and is most preferably methyl.

[0082]The term "aryl" as used herein, alone or in combination, refers to
an aromatic ring containing from 6-10 ring carbon atoms and up to a total
of 15 carbon atoms. It includes such groups as phenyl, α-naphthyl,
β-naphthyl and the like. The preferred aryl is phenyl. It excludes
heteroaryls.

[0083]Aralkyl groups are aryl groups attached to the main chain through an
alkylene bridge. Such groups include benzyl, phenethyl and the like.

[0084]"Lower alkyl carbonyl" refers to a lower alkyl group attached to the
main chain through a carbonyl. Similarly, "aryl carbonyl" refers to an
aryl group attached to the main chain through a carbonyl group.

[0085]"Lower cycloalkyl," as used herein refers to a cycloalkyl group
containing 3-10 carbon ring atoms and up to a total of 15 carbon atoms.
The cycloalkyl group may be monocyclic or bicyclic or tricyclic. Examples
include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, norbornyl, adamanyl, decalinyl, and the like. The preferred
lower cycloalkyl groups are cyclopentyl and cyclohexyl.

[0086]"Lower cycloalkyl lower alkyl" is a lower cycloalkyl group attached
to the main chain through an alkylene bridge. Such groups include
cyclohexylmethyl, cyclopentylethyl and the like.

[0087]"Cycloalkenyl" refers to a lower cycloalkyl group, as defined
herein, containing at least one double bond and up to a maximum of 6
carbon-carbon double bonds. It is not completely aromatic; but it may
include an aromatic moiety. It may contain one ring or two or more rings
fused together. The double bonds may be located in one ring or both
rings. One or more rings may be completely aromatic, while the remaining
rings, if any, in the structure may each be completely saturated or
contain 1 or 2 double bonds. It is to be noted however, that
cycloalkenyl, as described herein, excludes aryl. Examples include
cyclohexenyl, cyclooctenyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-indanyl, and
the like.

[0088]As used herein, an "electron donating group" shall designate a group
that will release or donate electrons more than hydrogen would if it
occupied the same position in the molecule. See, J. March, Advanced
Organic Chemistry, 3rd Ed., John Wiley & Sons p. 237 (1985). These
types of groups are well known in the art. Examples include lower
alkylamino, diloweralkylamino, amino, halo, aryl, lower alkoxy, lower
aralkoxy, aryloxy, mercapto, lower alkylthio, and the like. The preferred
electron donating groups are amino, hydroxy, lower alkoxy, lower
alkylamino and diloweralkylamino.

[0090]A "positively charged electron withdrawing group" is an electron
withdrawing group bearing a positive charge and forming a stable bond to
a N-hydroxide (N--O). These types of groups are well known in the art.
Examples include uronium groups,

##STR00009##

and the like, wherein R18, R19, R20, R21, R22,
R23, R24 and R25 are independently hydrogen, lower alkyl,
lower alkoxy lower alkyl or if the imino cation is formed, R18 and
R20 taken together with the nitrogen atoms to which they are
attached and the carbon atom therebetween may form a ring containing up
to 6 ring atoms and up to a total of 5 ring carbon atoms or R18 and
R19 taken together with the nitrogen atom to which they are attached
or R20 and R21 taken together with the carbon atoms to which
they are attached may form a 5 or 6-membered heterocyclic ring containing
up to a total of 5 ring carbon atoms or if the uronium cation is formed,
R20 and R18 may be taken with the nitrogen to which they are
attached form a 5 or 6 membered heterocyclic ring containing up to a
total of 5 ring carbon atoms or both R18 and R10 taken together
with the nitrogen atoms to which they are attached or R20 and
R21 taken together with the nitrogen atom to which they are
attached, may each simultaneously form a 5 or 6-membered heterocyclic
ring, each ring containing up to a total of 5 ring carbon atoms. In the
uronium and imino cations, it is preferred that R18 and R10 and
R20 and R21, when present, are the same. It is especially
preferred that R18, R19, R20, R21, whenever present,
are the same. It is also more preferred that the rings formed with
respect to the uronium, imino, and phosphonium cations are 5 or 6
membered rings.

[0091]With respect to the phosphonium cation, R18 and R19 and/or
R20 and R21 and/or R22 and R23 may each be
independently taken together with the nitrogen atoms to which they are
attached to form a ring. Thus, the phosphonium cation may be comprised of
1 ring, two rings or three rings. It is preferred that R18 and
R19, or R20 and R21 or R22 and R23 are the same.
It is especially preferred that R18, R19, R20, R21,
R22 and R23 are the same.

[0092]With respect to cations of Formula II, preferred cyclic uronium and
imino groups have the formula

##STR00010##

wherein R19 and R20 are as defined hereinabove and n1 and
n2 are independently 0 or 1, and U1 and U2 are
independently O, CH2 or NH or N-Alk wherein Alk is lower alkyl.

[0093]In the above formulae, the preferred values of R18, R19,
R20 R21, R22 and R23 are independently methyl, ethyl,
n-butyl, pentyl and --CH2CH2--O--CH2CH3. It is
preferred that R18, R19, R20 (for all) and R21, when
present (for imino), and R22 and R23 (for phosphonium) when
present, are the same.

[0094]The preferred values of R18, R19, R20, R21, when
present, and R22, and when present, and R23, when present, are
independently lower alkyl, especially methyl. It is preferred R18,
R19, R20 and R21, when present, R22, when present,
and R23, when present, are all the same. Further it is preferred
that all are methyl.

[0095]With respect to the uronium and/or imino cations, when R18 and
R19 or R20 and R21 taken together form a ring, they may
form heterocyclic moieties of the formula:

[0097]Preferred cyclic groups present in the phosphonium cations also have
the formula

##STR00012##

wherein U, is defined hereinabove.

[0098]It is preferred that R1 and R2 taken together with the
carbon atoms to which they are attached form an aryl or heteroaryl ring
wherein the aryl ring is a phenyl or naphthyl and the heteroaryl ring
contains 5 to 10 ring atoms and one or two ring heteroatoms consisting of
O, S, N and 3-8 ring carbon atoms.

[0099]With respect to compounds of Formula I, the following are preferred.
When n is 1, it is preferred that R7 and R8 are hydrogen or
lower alkyl, but most preferably hydrogen.

[0100]Preferred values of Y are S, O, NR4, or CR4R5,
wherein R4 and R5 are independently hydrogen or methyl.
Especially preferred values of Y are O, CH2 and NH. It is also
preferred, however, that Q is absent and Y forms a double bond with X.

[0101]It is preferred that X is CR6R7 or NR6. Preferred
values of R6 and R7 are hydrogen or lower alkyl.

[0102]With respect to compounds of Formula I, it is preferred that Q is
not present and Y and X are independently N or CH. It is more preferred
that at least one of Y and X is N and the other is CH. It is even more
preferred that Y is N and X is CH or N or that Y and X are both N.

[0103]When R6 and R7 taken together form an oxo group, X becomes
C═O. It is most preferred that X is C═O, CH2 or NH or
N(CH3). Moreover, an embodiment of Formula I has the formula

##STR00013##

[0104]In cases when n is 0, then R4 and R6 taken together may
form a bond between X and Y, i.e., a bond may form between the ring
carbon atom of X and the ring carbon atom of Y, or between the ring
nitrogen atom of X and the ring nitrogen atom of Y, or the ring nitrogen
atom of X, and the ring carbon atom of Y or the ring carbon atom of X and
the ring nitrogen atom of Y. In other words, under these circumstances
when n is O and R4 and R6 taken together form a bond between X
and Y, the compound of Formula I becomes

##STR00014##

[0105]wherein R1, R2, Y, X, and R3 are as defined above.
Under these circumstances, it is preferred that Y is CH or N and X is CH
or N. It is most preferred that Y and X are N.

[0108]As indicated hereinabove, when n is 1, R4 and R8 taken
together may form a bond between Q and Y, i.e., the ring carbon atom of
R4 and the ring carbon atom of R8 may form a bond, or the ring
carbon atom of R4 and the ring nitrogen atom of R8 may form a
bond, or the ring nitrogen atom of R4 and the ring carbon atom of
R8 may form a bond or the ring nitrogen atom of R4 and the ring
nitrogen atom of R8 may form a bond. For example, under these
circumstances, the compound of Formula I becomes:

##STR00016##

[0109]The preferred values of X in this formulations are C═O or NH or
CH2.

[0110]When n is 1, preferred values of Q are CH2 or NH. However, it
is also preferred that the compounds of Formula I have the formula:

##STR00017##

wherein [0111]Q is CR9 or N, and [0112]R9 is hydrogen or lower
alkyl and R1, R2, X, OR3 and Y are as defined hereinabove.
Examples of compounds of Formula I include:

##STR00018##

[0112]wherein R1, R2 and R2 are as defined hereinabove.

[0113]It is also preferred that compounds of Formula I have the formula:

##STR00019##

wherein R1, R2, R3, Y and X are as defined hereinabove.

[0114]The most preferred compounds of Formula I have the formula:

##STR00020##

or N-oxides thereofwherein Q, Y, X, R3, n, are as defined
hereinabove, [0115]A is N or CR24; [0116]D is CR25 or N;
[0117]E is CR26 or N; [0118]G is CR27 or N; and [0119]R24,
R25, R26 and R27 are independently hydrogen or lower alkyl
or an electron donating group or R25 and R26 or R24 and
R25 or R26 and R27 taken together form with the carbon
atom to which they are respectively attached an aryl ring. It is
preferred that A, D, E or G all are CH and more preferably at least one
of A, D, E, G is N.

[0120]It is preferred that no more than two of A, D, E, G are N. It is
most preferred that only one of A, D, E, G is N. Further it is preferred
that R24, R25, R26 or R27 are hydrogen or an
electron-donating group, as defined herein. The preferred electron
donating group is lower dialkylamino especially N,N-dimethyl amino and
lower alkoxy, e.g., methoxy.

[0121]Preferred compounds of Formula III have the formulae:

##STR00021##

or N-oxides thereofwherein Y, X, n, Q and R3 are as defined
hereinabove and R24 and R25 are independently lower alkyl,
hydrogen or an electron donating group.

[0122]Of the compounds of Formula IV-VII, when n is 1, the most preferred
compound is that of Formula IVa

##STR00022##

or N-oxides thereofwherein Q, Y, X and R3 are as defined hereinabove
and R24 and R25 are lower alkyl or hydrogen or an electron
donating group.

[0123]Preferred compounds of Formula I also have the formula

##STR00023##

or N-oxides thereofwherein n, Q, D, E, X and Y are as defined hereinabove
and J is NR28, O, CR28R29 or S(O)p, and p is 0, 1, 2.
[0124]R28 is hydrogen, lower alkyl or electron donating group as
defined hereinabove and R29 is hydrogen or lower alkyl. It is
preferred that R29 is hydrogen. The preferred values of R28 is
an electron donating group or hydrogen.

[0125]Preferred values of J are O or S(O)p; the preferred value of p is 1.

[0126]Preferred compounds of Formula VIII when n is 1 have the formula:

##STR00024##

or N-oxides thereofwherein J, Y, R8, R9, n and R3 are as
defined hereinabove and X is C═O.

[0127]In compounds VIII, IX, and VIIIa as depicted above, at least one of
D, E, or J is a heteroatom. Furthermore, it is most preferred that at
most two of J, E, and D are heteroatoms. It is most preferred that only
one of J, E, and D is a heteroatom.

[0128]When n is 0, preferred compounds of Formula I becomes:

##STR00025##

[0129]Thus, the present invention includes compounds having the formula:

[0130]The compounds of Formula I more preferably are compounds of the
formula:

##STR00027##

or N-oxides thereof.

[0131]In the above formulae, when the ring contains Y═X, this means
that R4 of Y and R6 of X are joined together to form a ring
bond between the Y ring atom and the X ring atom, so that as depicted
hereinabove there is a double bond between the Y ring atom and the X ring
atom.

[0132]Furthermore, in the above formulae, when the ring contains Y═N,
then R4 of Y and R8 of NR8 of Q join together to form a
ring bond so that there is a double bond between the nitrogen ring atom
and the Y atom. Thus, Y is CR5 or N under these circumstances.

[0133]The most preferred embodiment of Formula I has the formula:

##STR00028##

wherein one or two of A, D, E, G, is N and the rest are CH and X is CH or
N. It is most preferred that X is N. It is also preferred that at most
one of A, D, E and G is N and the rest are CH. It is most preferred that
A is N and especially G is N.

[0134]Preferred embodiments of compounds of Formula I include:

##STR00029##

or the N-oxides thereof.

[0135]With respect to compounds of Formula I, R3 is as defined
hereinabove. The various groups on R10, R11, R12 and
R13, e.g., alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heterocyclic, heterocyclic lower alkyl or lower cycloalkyl heterocyclics
may be unsubstituted, or substituted by lower alkyl or electron donating
or electron withdrawing groups. It is preferred that the groups are
unsubstituted or substituted by lower alkyl.

[0136]With respect to compounds of Formula I, it is preferred that
R10 is OR12, lower alkyl, aryl or aryl lower alkyl. It is more
preferred that R10 is OR12 or aryl.

[0137]It is also preferred that R11 is OR13, lower alkyl, aryl
or aryl lower alkyl. It is most preferred that R11 is OR13 or
aryl. Preferred values of R12 and R13 each independently are
lower alkyl, aryl or aryl or aryl lower alkyl. It is most preferred that
R12 and R13 are alkyl having 1-3 carbon atoms, or phenyl. It is
also preferred that R12 and R13 are the same.

[0138]It is also preferred that R10 and R11 are connected to
each other by a bridging group, T1. Preferred values of T1 are
O, CH2, S, or NR30 when R30 is lower alkyl and more
preferably H. When R10 and R11 are joined together, then
R3 becomes

##STR00030##

wherein R10, R11 and T, are as defined hereinabove. As defined
herein R3 may be defined as

##STR00031##

[0139]It is preferred that rings A1 and B are independently aromatic,
especially phenyl.

[0140]It is preferred that R3 is

##STR00032##

wherein R10' and R11', R12 and R13 are independently
lower alkyl, aryl or aryl lower alkyl and Rb1, Rc1, Rb2
and Rc2 are independently hydrogen or lower alkyl. It is also
preferred that R12 and R13 are connected by a bridging group
T1 to form the following R3 moiety

##STR00033##

[0141]In an embodiment of the present invention, R10 and R11 are
connected by the bridging group T1, as depicted hereinabove.
Preferred values of T1 are CH2, O, S and NH and most preferably
CH2 and O.

[0142]Embodiments of R3 include

##STR00034##

wherein R16 and R17 are independently lower alkyl, Rb1 and
Rc1 are independently H or lower alkyl and T is as defined
hereinabove and preferably O, NH or CH2.

[0143]Examples of R3 include

##STR00035##

[0144]Of course, various combinations and permutations of the formulae
described herein are also contemplated by the present invention. In
addition, Markush groupings containing less than all of the elements
described hereinabove as well as the various permutations thereof are
also contemplated by the present invention.

[0145]The compounds of Formula I can be prepared by art recognized
techniques. An illustrative technique is described hereinbelow.

[0146]For example, compounds of formula

##STR00036##

can be prepared by reacting

##STR00037##

under substitution reaction conditions wherein R1, R2, Y, Q, n,
R3, R10 and R11 are as defined hereinabove and L is a
leaving group, such as halo and the like.

[0147]It is preferable that the reaction is run in an inert polar organic
solvent and that the reactants are soluble therein at room temperature.
It is also preferred that the product of the substitution is insoluble in
the solvent at room temperature. Examples of suitable solvents for the
reaction include chloroform, carbon tetrachloride, ethyl ether, dioxane,
tetrahydrofuran and methylene dichloride, and the like. The reaction
takes place at effective temperatures, which may range from the melting
point of the solvent to reflux temperature thereof but it is preferred
that the reaction take place at about room temperature or at slightly
elevated temperatures up to the reflux temperature of the solvent. It is
especially preferred that the reaction take place at room temperature or
at slightly elevated temperatures, such as up to 60° C.

[0148]Compounds of Formula 100 can be prepared as described in U.S. Pat.
No. RE 37,686, RE 38,073, 5,580,981, 5,644,029, 5,698,675, the contents
of which are incorporated by reference.

[0149]For example, compounds of Formula 100, such as

##STR00038##

can be prepared by reacting hydrazine with

##STR00039##

respectively, wherein R' is halogen, NH--NH2 or OR'', and [0150]R''
is lower alkyl, such as methyl. This reaction is performed at slightly
elevated temperatures, such as 70-100° C., although the reaction
may be performed at temperatures ranging from room temperature to the
boiling point of the solvent.

[0151]The reaction is usually run in an organic solvent in which the
reactants are insoluble at room temperature, but in which the reactants
and product are soluble at slightly elevated temperatures. Examples of
useful solvents include ethanol, DMF and the like. In many cases, there
is a color change in the reaction mixture, indicating the formation of
the product. Work-up, such as removal of the solvent, followed by
acidification provides the desired product.

[0152]The hydrazino derivative (R'═NH--NH2) can be prepared by
reacting the corresponding halide, such as chloride or bromide, with
hydrazine under substitution reaction conditions. The ether derivative
(R1═OR'') can be prepare by reacting the corresponding alcohol
with an alkylating reagent, such as Me2SO4/Na2CO3,
under ether forming conditions.

[0153]Compounds of Formulae 100a, 100b, or 100c are useful for preparing
compounds of Formula I. These latter compounds can also be prepared by
art-recognized techniques. For example, compounds of Formula I are
prepared by reacting compounds of 100a, 100b, 100c, respectively, with

##STR00040##

under substitution reaction conditions wherein R10 and R11 are
defined hereinabove and L is a leaving group, such as halo, (e.g., Cl,
Br, I) and the like.

[0154]The N-oxides can be prepared from the compounds of Formula I having
a nitrogen ring heteroatom in the heteroaryl group. These N-oxides are
prepared by art-recognized techniques such as by oxidation thereof, such
as with peracid, e.g., peracetic acid or m-chloroperbenzoic acid.

[0155]With respect to compounds of Formula II, it is preferred that
Y1 is N or CR15, wherein R15 is hydrogen or methyl.
Especially preferred values of Y1 are CH and N.

[0156]It is also preferred that Qt is N or CR16 wherein R16
is hydrogen or lower alkyl. The preferred value of Q1 is N or CH.

[0157]With respect to compounds of Formula II, preferred values of Y1
and Q1 are CH or N. In a preferred embodiment, the compound of
Formula II has the formula

##STR00041##

wherein R1 and R2 and R14 are as defined as hereinabove and
Q1 is N or CH, but especially N.

[0158]Another embodiment of the compound having Formula II or salt of
Formula II, wherein the cation has the formula is:

##STR00042##

wherein [0159]R1 and R2 taken together with the carbon atoms
to which they are attached form an heteroaryl ring wherein said
heteroaryl ring is an oxygen, sulfur or nitrogen heteroaromatic
containing from 3 to 13 ring carbon atoms and 1-4 heteroatoms selected
from O, S and N, said heteroaryl ring may be unsubstituted or substituted
with lower alkyl or electron donating group; [0160]Y1 is N or
CR15; [0161]R15 is H or lower alkyl; [0162]Q1 is N or
CR16; [0163]R16 is H or lower alkyl; [0164]R14 is
hydrogen, a positively charged electron withdrawing group,

[0176]Another embodiment of the compound having Formula II or salt of
Formula II, wherein the cation has the formula is:

##STR00044##

[0177]or N-oxide or salt thereofwherein one of Y1 and Q1 is
CR15 and the other is N or CH; [0178]R15 is H or lower alkyl;
[0179]R1 and R2 taken together with the carbon atom to which
they are attached form an aryl or heteroaryl ring wherein said aryl ring
is an aromatic ring containing 6-14 ring carbon atoms and said heteroaryl
ring is an oxygen, sulfur or nitrogen heteroaromatic containing from 3 to
13 ring carbon atoms and 1-4 heteroatoms selected from O, S and N, said
heteroaryl ring may be unsubstituted or substituted with lower alkyl or
electron donating group;

[0192]As defined herein, in some embodiments of Formula II, R1 and
R2 taken together with the carbon atoms to which they are attached
form an aryl ring. It is preferred that the aryl ring is naphthyl and is
especially phenyl.

[0193]Moreover, in Formula II, as defined herein R14 is preferably H
or a positively charged electron withdrawing group, as defined
hereinabove, or

##STR00046##

wherein R10 and R11 are as defined herein and the electron
withdrawing group is as defined hereinabove. It is most preferred that
R14 is

##STR00047##

[0194]The preferred values of R10 and R11 are as defined
hereinabove with respect to Formula I.

[0195]Preferred structures of Formula II have the formula:

##STR00048##

wherein [0196]Y1 is N or CR15; [0197]R15 is H or lower
alkyl; [0198]Q1 is N or CR16; [0199]R14 is hydrogen, a
positively charged electron withdrawing group, [0200]R16 is H or
lower alkyl;

##STR00049##

[0200]SO2R17, lower alkyl carbonyl, aryl carbonyl, lower alkyl
aryl or BLK1-AA1 AA1 is an amino acid or peptide less a
hydrogen atom on the N-terminus and an OH on the C-terminus;
[0201]BLK1 is an amino protecting group; [0202]R10 is
OR12, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl, lower
cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lower
cycloalkenyl, or lower cycloalkenyl lower alkyl; [0203]R11 is
OR13, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl, lower
cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lower
cycloalkenyl or lower cycloalkenyl lower alkyl; [0204]and R10 and
R11 may optionally be connected by a bridging group selected from
the group consisting of O, S, NR30, or (CHR30)m, wherein
each R30 is independently lower alkyl or hydrogen and m is 1-3; and
[0205]R12 and R13 are independently lower alkyl, lower
cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocyclic
lower alkyl, lower cycloalkenyl or lower cycloalkenyl lower alkyl;
[0206]ring A1 and ring B are independently aromatic containing 6 to
14 ring carbon atoms or cycloalkenyl or cycloalkyl each containing 5 to
14 ring carbon atoms, and [0207]Rb1, Rc1, Rb2, Rc2
are independently hydrogen, lower alkyl or electron donating group and T
is CHR31, O, S or NR31 wherein R31 is hydrogen or lower
alkyl; [0208]A is N or CR24; [0209]D is N or CR25; [0210]E is N
or CR26; [0211]G is CR27 or N; [0212]R24, R25,
R26 and R27 are independently hydrogen or lower alkyl or an
electron donating group or R25 and R26 or R24 and R25
or R26 and R27 taken together with the carbon atoms to which
they are attached form an aryl ring, but at least one of A, D, E and G is
N.

[0213]It is also preferred that structures of Formula II have the formula:

##STR00050##

wherein [0214]Y1, Q1 and R14, are as defined hereinabove,
[0215]D is CR25 or N; [0216]G is CR26 or N; [0217]J is
NR28, O, CR28R29 or S(O)p; [0218]R25 and
R26 are independently hydrogen or lower alkyl or an electron
donating group or R25 and R26 taken together with the carbon
atoms to which they are attached form an aryl group; [0219]R28 is
hydrogen or electron donating group or lower alkyl; [0220]R29 is
hydrogen or lower alkyl and [0221]p is 0, 1 or 2.

[0222]More preferred structures of Formula II have the formula

##STR00051##

wherein [0223]A is N or CR24, [0224]D is CR26 or N, [0225]E is
CR26 or N. [0226]G is CR22 or N; [0227]J is NR28, O,
CR28, R29 or S(O)p; and [0228]R24, R25,
R26, R27, R28, R29, p, Q1, and R14 are as
defined hereinabove.

[0229]With respect to Formula II, and all of its various embodiments
depicted hereinabove, it is preferred that no more than two of A, D, E,
and G are N. It is most preferred that only one of A, D, E, or G is N.
Further, it is preferred that R24, R25, R26 or R27
are independently hydrogen or an electron donating group, as defined
herein. The preferred electron donating groups are lower dialkylamino,
especially N,N-dimethylamino and lower alkoxy, e.g., methoxy.

[0230]Moreover, it is preferred that T is CH2, O, S or NH and more
preferably CH2 or O. Preferred structures of Formula X have the
formulae

##STR00052##

or N-oxides thereof, wherein R24, R25, Q1 and R14 are
as defined hereinabove.

[0231]Of the structures of Formulae XI-XIV, the most preferred compound is
that of XIa viz.

##STR00053##

wherein R24, R25, Q1 and OR44 are as defined herein.

[0232]Preferred structures of Formula II also have the formula

##STR00054##

wherein J, E, D, Q1 and R14 are as defined hereinabove. It is
preferred that R24 is hydrogen, lower alkyl or electron donating
group as defined hereinabove and R25 is hydrogen or lower alkyl. It
is most preferred that R25 is hydrogen and it is most preferred that
R24 is an electron donating group or hydrogen.

[0233]Preferred values of J are O or S(O)p or NH; the preferred value
of p is 1.

[0234]Preferred structures of Formula II also have the following formula:

##STR00055##

or the N-oxides thereof.

[0235]In the various structures described herein whether it is for
compounds of Formula I or II or any other embodiment of the present
invention depicted herein, the preferred values of T is O, S, NH or
CH2. In addition, in the various structures depicted hereinabove, it
is preferred that m is 1 and that R30 is H.

[0236]Compounds of Formula II or salts, especially wherein the cationic
portion has the structure of Formula II, are prepared by art recognized
techniques For example compounds of Formula II are prepared by reacting
compounds of Formula XVII whenever R14 is hydrogen

##STR00056##

[0237]with R14L, wherein R1, R2, Q1, and R14 is
as defined herein and L is a leaving group, such as halo, (e.g. chloro,
bromo or iodo). However, if R14 is a positively charged electron
withdrawing group, then the structures of Formula II is a cation, and in
this case, there would be an anion associated with this cationic moiety.
For example, when R14 is

##STR00057## the compounds of Formula XVII is reacted with

##STR00058## under substitution reaction conditions wherein R12
and R11 and L are as defined hereinabove. It is preferable that
reaction is run in an inert polar organic solvent and that the reactants
are soluble therein at room temperature. It is also preferred that the
product is insoluble in the solvent at room temperature Examples of the
solvent that could be used include chloroform, carbon tetrachloride,
ethyl ether, dioxane, tetrahydrofuran, methylene, chloride, and the like.
The reaction takes place at effective temperatures, which may range from
the melting point of the solvent up to reflux temperatures, but it is
preferred that the reaction takes place at about room temperature or at
slightly higher temperatures up to the reflux temperature of the solvent.
It is especially preferred that the reaction take place at room
temperature or at slightly elevated temperatures such as up to 60°
C.

[0238]Compounds of Formula XVII can also be prepared by art recognized
techniques known to one of ordinary skill in the art. An exemplary
procedure is as follows:

##STR00059##

[0239]For example, a compound of Formula VII is refluxed with acetic
anhydride to give the corresponding anhydride under anhydride formation
conditions. The anhydride product was then treated with acetamide under
amide forming conditions to give the corresponding cyclic imide XIX. The
cyclic imide XIX is subjected to conditions effective for it to undergo
Hoffman rearrangement, e.g., by reacting it with sodium hypobromite (or
sodium hydroxide and bromine) followed by hydrolysis. For example, the
cyclic imide XIX is reacted with NaOBr, Copper (II) acetate and H2S
to provide the amino carboxylic acid (XX) with one less carbon atom.
Esterification of acid XX under the esterfying conditions gives the
corresponding ester XXI. Treatment of XXI with hydroxylamine in acid
(hydroxylammonium salt) under esterification reaction conditions gives
the hydroxamic acid derivative XXII. Diazotization followed by
intramolecular cyclization gives the azo derivatives XXIII. On the other
hand, reaction of the hydroxamic with formic acid under effective
conditions, such as by heating the hydroxamic acid with formic acid at
effective temperatures e.g., temperatures ranging from just above room
temperature up to and including reflux temperatures, and preferably, at
the reflux temperature of formic acid gives the product XXIV.

[0240]Of course, various combinations and permutations of the formulae
described herein are also contemplated by the present invention. In
addition, Markush groupings containing less than all of the elements
described hereinabove as well as the various permutations thereof are
also contemplated by the present invention.

[0241]As described herein, the compounds or salts or N-oxides described
hereinabove are useful in promoting peptide coupling, i.e., the reaction
between a free amino group of a first amino acid or first peptide with a
free carboxy group or acylating group of a second amino acid or peptide.
The process of the present invention is general; it can be used in
effecting the coupling of a dipeptide of an amino acid, a tripeptide and
an amino acid, dipeptides, pentapeptide, higher peptides, polypeptides,
etc.

[0242]When the compound of Formula I or structures of Formula II reacts
with an amino compound such as an amino blocked amino acid or protein of
the Formula BLK1-AA1, the corresponding amino acid ester of the
one of the following compounds is formed depending on the identity of the
coupling agent:

##STR00060##

wherein AA1 is an amino acid or protein as defined herein, BLK1
is a blocking group as defined herein and Y, Q, Q1, n, X, R1,
and R2 are as defined hereinabove. This amino acid ester can then
react with a compound having a free amino group such as an arylamino,
alkylamino, lower aryl amino, heterocyclic amino, heterocyclic lower
alkylamino, lower cycloalkylamino, lower cycloalkyl lower alkyl amino,
and the like designated as R33R34NH, wherein R33 and
R34 are independently hydrogen, lower alkyl, aryl or lower aryl
alkyl, to form a compound of the formula:

##STR00061##

[0243]Removal of the blocking group by techniques known to one skilled in
the art affords the product:

AA1NR33R34

[0244]This technique is extremely useful when the second amino compound is
an amino acid or peptide having a free amine group, designated as
AA2. For example, if the coupling agent is a compound of Formula I,
a peptide may be formed between AA1 and AA2 as follows, for
example,

##STR00062##

wherein AAl1, AA2, BLK1, R1, R, Y, Q, n and X are as
defined herein.

[0245]If the coupling agent contains a structure of Formula II,

##STR00063##

whether compound or cationic portion of the salt, then the reaction
becomes

[0247]The blocking group can be any of the blocking groups known in the
art but the preferred blocking groups are FMOC, BOC, benzyloxycarbonyl
BSMOC and Bspoc.

[0248]The term "amino acid" or AA, AA1, or AA2 as used herein
refers to an organic acid containing both a basic amino group (NH2)
and an acidic carboxyl group. (COOH).

[0249]Therefore, said molecule is amphoteric and exists in aqueous
solution as dipole ions. (See "The Condensed Chemical Dictionary",
10th Ed., edited by Gessner G. Hawley, Van Nostrand Reinhold
Company, London, England p. 48 (1981). The preferred amino acids are the
a-amino acids. They include but are not limited to the 25 amino acids
that have been established as protein constituents. They must contain at
least one carboxyl group and one primary or secondary amino group in the
amino acid molecule. The term includes such proteinogenic amino acids as
alanine, valine, leucine, isoleucine, norleucine, proline,
hydroxyproline, phenylalanine, tryptophan, 2,4-diamino butyric acid,
methionine, glycine, serine, threonine, cysteine, cystine, glutamic acid,
lysine, hydroxylysine, ornithine, arginine, histidine, penicillamine,
naphthylamine, α-phenylglycine, aspartic acid, asparagines,
glutamine, arginine, tyrosine, and the like.

[0250]As used herein, the term "peptide" refers to the class of compounds
composed of amino acid units chemically bound together with amide
linkages. A peptide may contain as little as two amino acid residues or
may consist of a polymer of amino acid residues (polypeptide).

[0251]As used herein, the terms "amino acid" and "peptide" also include
amino acids and peptides, respectively containing blocking (protecting)
groups. These protecting "groups" block the amino group or the carboxyl
group of the amino acid or peptide not involved in or taking part in the
coupling in order to prevent unwanted side reactions. These protecting
groups also protect reactive groups on the side chain.

[0252]A number of blocking reagents for amino groups are known in the art
and have been utilized in the syntheses of peptides. These blocking
groups are discussed in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396,
4,581,167, 4,394,519, 4,460,501 and 4,108,846, the contents of all of
which are incorporated by reference as if fully set forth herein. Other
amino protecting groups are discussed in U.S. Pat. Nos. 5,221,754,
5,510,491 and 5,637,719 the contents of which are also incorporated by
reference. Other amino protecting groups are described in an article
entitled "Solid Phase Peptide Synthesis," by G. Barany and R. B.
Merrifield in THE PEPTIDES, Vol. 2, edited by E. Gross and J.
Meienhoffer, Academic Press, N.Y., N.Y. 100-118 (1980), and in the book
entitled "PROTECTIVE GROUPS IN ORGANIC SYNTHESIS" by T. W. Green,

John Wiley & Sons, New York, the contents of all of which are being
incorporated by reference.

[0253]The term amino acid protecting group, (BLK, BLK1) as used
herein, refers to blocking groups which are known in the art and which
have been utilized to block the amino NH2) group of the amino acid.
Blocking groups such as 9-lower alkyl-9-fluorenyloxycarbony,
2-chloro-1-indanylmethoxy-carbonyl (CLIMOC) and
benz[f]indene-3-methyloxycarbonyl (BIMOC) and dbd-TMOC are discussed in
U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396, 4,581,167, 4,394,519,
4,460,501 and 4,108,846 referred to hereinabove, the contents of which
are incorporated by reference. Moreover, other amino protecting groups
such as 2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc) and
benzothiophene sulfone-2-methoxycarbonyl (Bsmoc). Other N-amino
protecting groups include such groups as the t-butyloxycarbonyl (BOC),
t-amyloxycarbonyl (Aoc), (3-trimethylsilylethyloxycarbonyl(TEOC),
adamantyl-oxycarbonyl (Adoc), 1-methylcyclobutyloxycarbonyl (Mcb),
2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc),
2-(p-phenylazophenyl)propyl-2-oxycarbonyl (Azoc),
2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz),
2-phenylpropyl-2-oxycarbonyl (Poc), bnzyloxycarbonyl (Cbz),
p-toluenesulfonyl aminocarbonyl (Tac), o-nitrophenylsulfenyl (Nps),
dithiasuccinoyl (Dts), Phthaloyl, piperidine-oxycarbonyl, formyl,
trifluoroacetyl and the like.

[0254]These protecting groups can be placed into four categories:

[0255]1) a base labile Na-amino acid protecting group such as FMOC, and
the like. [0256]2) protecting groups removed by acid, such as Boc, TEOC,
Aoc, Adoc, Mcb, Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl
(Foc), p-methoxybenzyloxycarbonyl (Moz), Nps, and the like. [0257]3)
protecting groups removed by hydrogenation such as Dts, Cbz. [0258]4)
protecting groups removed by nucleophiles, such as Bspoc, Bsmoc and Nps
and the like. [0259]5) protecting groups derived from carboxylic acids,
such as formyl, acetyl, trifluoroacetyl and the like, which are removed
by acid, base or nucleophiles.

[0260]A variety of carboxy protecting groups known in the art may be
employed. Examples of many of these possible groups may be found in
"Protective Groups in Organic Synthesis," by T. W. Green, John Wiley &
Sons, 1981, the contents of which are incorporated by reference. These
examples include such groups as methyl ester, t-butyl ester,
β-trimethylsilylethyl ester, benzyl ester and the like.

[0261]In addition, during the course of protein synthesis, it may be
necessary to protect certain side chains of the amino acids to prevent
unwanted side reactions. The various protecting groups are discussed in
U.S. Pat. No. 5,360,920, the contents of which are incorporated herein by
reference.

[0262]The term "acylating group of an amino acid or peptide" refers to a
group on the free carboxy end of the amino acid or peptide that
facilitates the acylation reaction, i.e., nucleophilic substitution at
the acyl carbon. Examples include the free acid, acid halide, esters,
such as lower alkyl esters, phenoxy esters which are unsubstituted or
substituted with 1-5 electron withdrawing groups as defined herein; or an
anhydride and the like. The preferred acylating derivative is the acid,
acid halide, especially the acid chloride or fluoride, and the phenoxy
ester.

[0263]The preferred acylating amino acid is an amino acid group of the
formula

BLK-AA-M,

wherein BLK is an amino protecting group [0264]AA is an amino acid less
the H on the COOH moiety and M is halo or

##STR00065##

[0264]wherein Ra is independently halo, lower alkyl, nitro, cyano or other
electron withdrawing group and b is 0-5. When b is 0, the phenyl group is
unsubstituted.

[0265]The most preferred acylating group of an amino acid is the amino
acid chloride or fluoride. The preparation and use of amino acid
chlorides as an acylating derivative is discussed in an article by
Camino, et al. in J. Org. Chem., 1986, 51, 3734-3736, the contents of
which are incorporated herein by reference. Briefly, amino acid chlorides
can be prepared by reacting the amino acid with thionyl chloride and
recrystallizing the product from a recrystallization reagent, such as
CH2Cl2-hexane.

[0266]The preparation and use of amino acid fluorides in peptide synthesis
are discussed U.S. Pat. No. 5,360,920, the contents of which are
incorporated herein by reference. As described therein, the amino acid
fluorides can be prepared by reacting an N-protected amino acid with the
reagent cyanuric fluoride. This reaction can be run at temperatures as
low as 0° C. and up to the refluxing temperature of the solvent,
but it is preferred that the reaction is run at room temperature. It can
also be run in an inert solvent, such as pyridine/CH2Cl2 and
the like. The cyanuric fluoride can be prepared from the corresponding
chloride in the presence of potassium fluoride at elevated temperatures
ranging from 150° to 250° C., according to the following
equation

##STR00066##

[0267]A typical preparation of the peptide in accordance with the present
invention involves the following steps

[0268]1) protection of the free carboxyl group in a first amino acid or a
first peptide, unless the amino acid or peptide is anchored to a solid
support.

[0269]2) protection of the free amino group of a second amino acid or
peptide.

[0270]3) protection of the side chains, if necessary.

[0271]4) coupling the first amino acid or peptide with the second amino
acid or peptide in the presence of compounds of Formula I.

[0272]5) removal of the protecting groups.

[0273]The procedure of steps 1-3 can be performed in any order.

[0274]In the coupling step, the compounds of Formula I or II or salts or
N-oxides thereof or cation of Formula II is present in effective amounts.
Usually, the first amino acid or peptide is present in approximately
equimolar amounts with the second amino acid or peptide. Furthermore, the
amount of the compound having Formula I or II used depends upon the
amount of peptide or amino acid which is present in the least amount
(i.e. the limiting reagent); thus the molar ratio of the compound of
Formula I or II to the amino acid or peptide present in the least molar
amount, ranges from about 1:3 to about 3:1, although it is preferred that
approximately equimolar amounts of the compound of Formula I or II (or
salt or N-oxide thereof or cation of Formula II) the first amino acid or
peptide and the second amino acid or peptide be used.

[0275]The coupling reaction usually takes place in an inert organic
solvent such as dimethylformamide (DMF) or ethers, such as ethyl ether,
THF or dioxane. In fact DMF is the preferred solvent in the solid phase
synthesis because of its favorable solvation properties. The reaction
takes place under mild conditions usually ranging from about 0° C.
to about 30° C. After the peptide is formed, the blocking groups
are removed by techniques known to one skilled in the art.

[0276]The following sequence is illustrative of the coupling reaction; in
the examples below, amino acids (AA) are used, although the procedure is
general for amino acids and/or peptides:

##STR00067##

[0277]In the above scheme, BLK is an amino acid blocking group, AA1,
AA2 and AA3 are first, second and third amino acid respectively
and P is a carboxy protecting group.

[0278]As shown by the above scheme, the N-α amino protected amino
acid is reacted with a second amino acid in which the carboxy group is
protected.

[0279]A peptide is formed between the first amino acid and the second
amino acid. The peptide chain can be increased by removing the alpha
amino protecting group by techniques known to one skilled in the art and
then reacting the corresponding dipeptide with another N-α amino
protected amino acid in the presence of a compound of Formula Ito form
the corresponding tri-peptide. The N-α amino protecting group of
the tri-peptide is removed and the above cycle is repeated until the
desired peptide has been obtained.

[0280]The present invention can readily be utilized in solid phase peptide
synthesis. Solid phase peptide synthesis is based on the stepwise
assembly of a peptide chain while it is attached at one end to a solid
support or solid phase peptide resin. Two methods are generally well
known in the art.

[0281]One, the Merrifield method, employs a solid support for attachment
of the amino acid or peptide residues. This method employs N-protected
amino acids as building blocks which are added to an amino acid or
peptide residue attached to the solid support at the acyl (acid) end of
the molecule. After the peptide bond has been formed, the protecting
group is removed and the cycle repeated. When a peptide having the
desired sequence has been synthesized, it is then removed from the
support.

[0282]The second method, the inverse Merrifield method, employs reagents
attached to solid supports in a series of columns. The amino acid or
peptide residue is passed through these columns in a series to form the
desired amino acid sequence.

[0283]These methods are well known in the art as discussed in U.S. Pat.
Nos. 4,108,846, 3,839,396, 3,835,175, 4,508,657, 4,623,484, 4,575,541,
4,581,167, 4,394,519 as well as in Advances in Enzymology, 32, 221 (1969)
and in PEPTIDES, Vol. 2, edited by Erhard Gross and Johannes Meienhoffer,
Academic Press, New York pp. 3-255 (1980) and the contents thereof are
incorporated herein by reference as if fully set forth herein.

[0284]The compounds of the present invention are useful as coupling agents
or bases in peptide coupling. However, their effectiveness is also a
function of the solvent system which is used to dissolve the reactants
and bases used in the coupling reaction. If the compound of the present
invention is not too soluble in the solvent used in the coupling method,
then it should be converted to a form more soluble in the solvent or the
solvent should be changed to one in which it is soluble. This is usually
not a problem in peptide coupling conducted in solution. But, with some
peptide synthesizers, the option of changing the solvent is not
available. For example, with some synthesizers, DMF or
N-methylpyrrolidone is the solvent utilized. However, compounds of the
present invention may not be too soluble in the solvent utilized, such as
DMF or N-methylpyrrolidone. Thus, to overcome this problem the compound
of the present invention is converted to a compound which is soluble in
the solvent utilized, such as DMF or N-methylpyrrolidone. For example, if
the coupling agent or base of the present invention is that of Formula I
and if R10 or R11 is aryl or heteroaryl or if A1 or B is
aryl or heteroaryl, then the trick to overcome this problem is to place a
t-butyl or t-amyl group or any other group wherein the carbon atom
attached to the aryl or heteroaryl ring is a tertiary carbon.

[0285]If R10 or R11 or A1 or B contain more than one ring
it is preferred that the t-butyl or amyl group or other tertiary carbon
group, such as a tertiary hydrocarbyl group (containing only carbon and
hydrogen atoms) is attached to the ring attached to the phosphorus atom.
If more than one such group is attached to the rings, it is preferred
that these groups are the same.

[0286]The following examples further illustrate the present invention.

[0335]A. Quinolinimide. 2,3-Pyridinedicarboxylic acid (22.5 g, 0.135 mol)
was added to 25 ml of acetic anhydride and the mixture was heated with
stirring to reflux on a steam-bath. A clear colorless solution was
obtained after about 1 hour, and the heating was continued for 2 more
hours. Then, acetic acid and excess acetic anhydride were distilled off
(˜12.6 g) until the temperature of the residual solution reached
165° C. The residue which remained in the flask solidified to a
dust red solid upon cooling to room temperature.

[0336]To the above solid, acetamide (14 g, 0.23 7 mol, 1.75 equiv) was
added and the mixture was heated overnight by means of an oil-bath,
keeping the temperature at 120-125° C. After cooling to room
temperature, the solid was collected and washed with acetic acid
(2×10 ml) and water (3×50 ml). The gray solid was suspended
in 250 ml of hot water and the mixture stirred for 15 min to give by
filtration 15 g (75%) of the above-identified imide as a gray solid: mp
237°-239° C. (lit. mp 233° C.).

[0337]B. 3-Aminopicolinic Acid. Quinolinimide (50 g, 0.344 mol) was
dissolved in 1000 ml of 10% NaOH while cooling in an ice-bath and to the
solution was added slowly with stirring and cooling a cold NaOBr
solution, which had been obtained by mixing 56 g (18 ml, 0.344 mmol) of
Br2 with 350 ml of 15% NaOH in an ice-bath. After the addition had
been completed, stirring was continued in the ice-bath for 15 min and at
room temperature for 1 hour. The resulting mixture was then heated to
85° C. with stirring for 1 hour. After cooling, the mixture was
neutralized to pH 5-6 by means of 50% H2SO4 and the whole was
kept at room temperature overnight.

[0338]The resulting white cloudy mixture was heated and the clear solution
treated with a hot solution of 22 g (0.11 mol) of copper (II) acetate
monohydrate in 400 ml of H2O and 10 ml of acetic acid. The mixture
was heated with a steam bath for 15 min and then cooled at room
temperature and the copper salt collected and washed twice with cold
water.

[0339]The copper salt was re-suspended in 500 ml of water, and H2S
was passed through the suspension for 2 to 3 hours while stirring. Black
CuS was removed from the mixture by filtration and the filtrate
decolorized with charcoal. Removal of water gave a dust yellow solid,
which was recrystallized form water-ethanol (1:1 v/v) to give 29 g (60%)
of the above-identified acid as a cream-yellow solid: mp 212-214°
C. (lit. mp 210° C.).

[0361]To a mixture of 0.4923 g (3 mmol) of HODhat as prepared in Example 4
and 0.46 ml (3.3 mmol) of TEA in 25 ml of dry CH2Cl2 at
0° C., 1.0 g (3 mmol) of PyClu was added portion wise with
stirring under an atmosphere of dry N2. Stirring was continued for 1
hour in an ice-bath and then at room temperature overnight. The clear
light yellow mixture was diluted with CH2Cl2 to 50 ml and
washed with ice cold water (2×15 ml) and dried over MgSO4. The
solvent was removed, and the oily residue was dissolved in 5 ml of MeCN
to which 30 ml of ether was added, and the whole was stored at
-20° C. for several days until the oil solidified. The solid was
collected by filtration and redissolved in 20 ml of CH2Cl2, and
the solution washed with ice cold water (2×5 ml) and dried over
MgSO4. Removal of solvent gave a pink-yellow solid which was
recrystallized from MeCN-ether to give 0.42 g (3 0%) of the
above-identified uronium salt product as white crystals: mp 136.5°
C. (explodes); 1H NMR (CD3CN) δ9.17 (dd, 1H), 8.67 (dd,
1H), 8.11 (dd, 1H), 3.75 (t, 8H, 4NCH2), 1.97 (m, 8H, 4CH2); IR
(KBr): 2985 (m), 1734 (vs, CON), 1679 (vs), 1448 (sh, s), 1341 (m), 1169
(m), 1072 (m), 964 (m), 846 (sh, vs) cm-1.

[0366]Method A. To a suspension of 0.42 g (3.054 mmol) of HOAt in 20 mL of
anhydrous CH2Cl2, there was added 0.43 mL (1 equiv.) of
triethylamine with magnetic stirring. The resulting clear yellow solution
was cooled in an ice bath under an atmosphere of N2 and treated
slowly with 0.85 g (1 equiv.) of 2,8-dimethylphenoxaphosphinic chloride.

[0368]Method B: To a suspension of 1.25 g of HOAt in 20 mL of anhydrous
CH2Cl2 there was added 0.623 g (1 equiv.) of imidazole with
magnetic stirring. The resulting white suspension was cooled in an ice
bath under an atmosphere of N2 and treated slowly with 2.56 g (1
equiv.) of 2,8-dimethylphenoxaphosphinic chloride. The reaction mixture
was stirred at 0° C. for 30 min, then at room temperature for 2
hours and diluted with 30 mL of CH2Cl2. The reaction mixture
was filtered in a sintered glass funnel over anhydrous MgSO4 under
an atmosphere of N2. After removal of solvent with a rotary
evaporator with the aid of a water aspirator, the residue was
recrystallized from CH2Cl2-hexane to give 2.86 g (82.3%) of the
above-identified phosphinic ester as white crystals, for which the mp and
NMR data agreed with the data reported above.

Example 14

[0369]Using the procedure described hereinabove, the following are also
prepared:

##STR00071##

[0370]2. OAt derivatives of

##STR00072##

wherein the phenoxophosphinic acid products have the following
substituents therein.

[0372]Using method B of Example 14, and using 4-Me-HOAt instead of HOAt,
the above-identified compound was made as a white solid.

Example 16

Di-o-tolyl-phosphinyloxy-7-azabenzotriazole (DtpOAt)

[0373]A. Di-o-toylphosphine oxide. Magnesium turnings (13.96 g) were added
to 100 ml of anhydrous ether in a 3-neck flask fitted with a condenser,
magnetic stirrer and a dropping funnel kept under an atmosphere of
nitrogen. o-Bromotoluene (100 g, 0.579 mol) in 100 ml of ether was slowly
added to the mixture. During the addition, the Grignard reaction was
initiated and became so vigorous that ice bath cooling was needed
frequently. After the addition was complete (40 min), the reaction
mixture was refluxed for 15 min and then cooled with an ice bath and
treated slowly with 30.8 ml (0.232 mol) of diethyl phosphite in 40 ml of
ether. The mixture was refluxed again for 15 min and cooled with an ice
bath. Two hundred and fifty milliliters of 10% HCl and 200 ml of water
were added slowly to the cooled mixture with magnetic stirring. Ether was
evaporated and the insoluble phosphine oxide was collected by filtration
and recrystallized from CH2Cl2-hexane (a few drops of methanol
may be added to help dissolve the solid) to give 39.47 g (73.9%) of the
phosphine oxide as a pale yellow solid: mp 94° C., lit. mp
93-94° C., yield 57%; 1H-NMR (60 MHz, CDCl3):
δ2.376 (s, 6), 4.234 (s, 1), 7.19-7.94 (m, 8); IR (KBr): 2369
(P--H), 1168 (P═O) cm-1.

[0374]B. Di-o-toylphosphinic acid. A suspension of 15.04 g of
di-o-toylphosphine oxide in 80 ml of 5N aqueous NaOH was treated with 40
ml of 30% H2O2 all at once and the resulting mixture was heated
on a steam-bath for 20 min. A clear solution resulted and was filtered
while hot. The filtrate was cooled in an ice bath and acidified slowly
with concentrated HCl, which caused the precipitation of a white solid
which was recrystallized from MeOH-ether to give 13.4 g (83.3%) of the
phosphinic acid: mp 174-176° C., lit. 175-177° C., yield
58-74%; 1H-NMR (60 MHz, TFA): δ2.369 (s, 6), 7.245-8.124 (m,
8); IR (KBr): 1143 (P═O) cm-1.

[0376]The above-identified compound was prepared according to the
following scheme which is a variation of the procedure of Example 16:

##STR00074##

[0377]A. Preparation of Di-o-tolylphosphine oxide (1) Magnesium turnings
(13.96 g) were added to 100 mL of anhydrous ether under a nitrogen
atmosphere in a 1 L 3-neck flask fitted with an efficient condenser,
magnetic stirrer and two dropping funnels. o-Bromotoluene (72 mL) was
added to the mixture. The rate of addition was adjusted to allow the
ether to boil slowly. No ice bath cooling was necessary during addition.
After the addition was completed the reaction mixture was refluxed for 2
hours and then cooled in an ice bath. Diethyl phosphite (30.8 mL) in 50
mL of ethyl ether was added from the second dropping funnel and the
reaction mixture was stirred at room temperature overnight. Diethyl
phosphite was distilled before use, by 62-64° C. (5 mm Hg). Two
hundred and fifty milliliters of 10% HCl and 200 mL of water were added
slowly to the cooled mixture and the ether was removed with a repave. The
insoluble material was isolated by filtration, dried and recrystallized
from toluene to give 30.7 g (57.4%) of the phosphine oxide as pale yellow
crystals, mp 95-97° C., lit. mp 94-95° C.

[0378]B. Preparation of di-o-tolylphosphinic acid (2)-Di-o-tolylphosphine
oxide (15 g) and 80 mL of 5 N NaOH were treated with 40 mL of 30%
H2O2. The suspension was heated gently on a steam bath for 20
min. The heating was conducted carefully because the reaction could
potentially be violent. Carrying out the reaction in a large beaker
avoided loss of product if the reaction became violent. (The reaction
mixture doesn't explode but a lot of foam and gas are developed.) The hot
reaction mixture was added to an iced HCl solution (90 mL of conc. HCl
plus 30 g of ice). The resulting white solid was isolated by extraction
with chloroform (3×75 mL). The chloroform layer was washed with
water and dried over magnesium sulfate. After the solvent was removed in
vacuum the resulting solid was recrystallized from 95% ethanol. The above
amount of phosphine oxide was oxidized in two batches of 15 g each. The
average yield of the two runs was 79% for the pure phosphinic acid, mp
174-175° C., lit. mp 175-177° C.

[0379]C. Preparation of di-o-tolylphosphinic acid chloride (3).
Di-o-tolylphosphinic acid (22.5 g) was added in one portion to 75 mL of
thionyl chloride and the reaction mixture heated under reflux for 3
hours. After removal of excess thionyl chloride, the residual oil was
dissolved in dry dichloromethane (DCM), and the DCM removed at the
rotavap, the operation being repeated three times. The acid chloride
which was obtained as an oil which solidified upon standing was used
without further purification.

[0380]D. Preparation of Dtp-OAt- To a suspension of 12.7 g of HOAt in 300
mL of anhydrous DCM was added 16.7 mL of DIEA. The resulting clear yellow
solution was cooled in an ice bath under an atmosphere of nitrogen and
treated with 25.8 g of di-o-tolylphosphinic acid chloride dissolved in
300 mL of DCM. The reaction mixture was stirred at 0° C. for 1
hour and then at room temperature for 2 hours. The reaction mixture was
diluted with 600 mL of DCM and washed with water and saturated aqueous
NaCl and dried over MgSO4. After removal of solvent with a rotary
evaporator with the aid of a water aspirator, the residue was
recrystallized from CH2Cl2/ethyl acetate to give 30.1 g (88%)
of the active ester as white crystals, mp 175-177° C., lit. mp
170-172° C.

[0381]It was noted that this material has very low solubility in DMF. Some
automatic peptide synthesizes, for example, the MB 433 A peptide
synthesizer, requires a solution of the activator in DMF to be placed in
one of the reagent bottles which then delivers the activated species to
the reaction vessel. The concentration of the reagent in solution has to
be in the range of 0.3-0.8 M to guarantee the right concentration of the
active species. Since the solubility of this reagent is so low in DMF,
the yield for coupling two amino acids to form a peptide was low if this
automatic synthesizer was used. However, a slight modification of the
structure of the product of this example to make it soluble in DMF
enhances its ability to couple amino acids to form peptides. The
following example illustrates a structural modification of Dtp-OAt which
dramatically enhances its solubility in DMF and other organic solvents.

Example 18

5-t-Bu-Dtp-Oat

[0382]The new coupling reagent was synthesized according to the strategy
outlined hereinbelow. Starting from the commercially available
4-t-Bu-toluene the synthesis is completed in 5 steps.

##STR00075##

[0383]A. Preparation of 2-bromo-4-t-butyltoluene (4). The bromination was
carried out according to the procedure of Reich, et al. J. Med. Chem.
1996, 39, 2781, the contents of which are incorporated by reference. To a
solution of 4-t-butyltoluene (90 mL, 0.518 mol) and two crystals of
I2 was added dropwise bromine (27 mL, 0.524 mol). The reaction
mixture was stirred at room temperature for 3 hours and then poured into
1 L of cold water. The mixture was transferred to a separatory funnel and
the lower layer was collected. The crude 2-bromo-4-t-butyltoluene was
washed with saturated NaHCO3, brine and dried over MgSO4. The
crude product was distilled under reduced pressure, first using a water
aspirator and then with a high vacuum oil pump. A first fraction was
collected up to b.p. 100° C. with the aid of the water aspirator
and the residue was distilled in high vacuum to give 90.5 g (77%) of the
bromide as a colorless liquid, by 61-65° C. (3-5 mm/Hg).

[0386]D. Preparation of bis-(5-t-butyl-2-methylphenyl)phosphinic chloride
(7). The preparation was carried out as described for compound (3) as
described in Example 17. The acid chloride was used in the next step
without purification. 1H-NMR (400 MHz, CDCl3) δ1.276
(18H, s), 2.384 (6H, s), 7.173-7.213 (2H, m), 7.493 (2H dt), 7.903 (2H,
dd), IR (KBr) 1231 cm-1 (P═O).

[0387]E. Preparation of
bis-(5-t-butyl-2-methylphenyl)phosphinyloxy-7-azabenzotriazole. To a
suspension of 0.76 g (5.58 mmol) of HOAt in 30 mL of anhydrous DCM was
added 0.78 mL (5.6 mmol) of TEA. The resulting clear yellow solution was
cooled in an ice bath under an atmosphere of nitrogen and treated with 2
g (5.27 mmol) of bis-(5-t-butyl-2-methylphenyl)-phosphinic acid chloride
dissolved in 20 mL of DCM. The reaction mixture was stirred at 0°
C. for 1 hour and then at room temperature for 4-5 hours. The reaction
mixture was diluted with 40 mL of DCM and washed with water and saturated
aqueous NaCl and dried over MgSO4. The solvent was removed with a
rotary evaporator with the aid of a water aspirator. The above product
was obtained as a white solid [2.3 g (86%)] after recrystallization from
hexane and then from hexane containing a small amount of ethyl acetate:
mp 86-87° C., 1H-NMR (400 MHz, CDCL3) δ1.271 (18H,
s), 2.687 (6H, s), 7.264-7.298 (2H, m), 7.369 (1H, dd), 7.545 (2H dt),
7.974 (2H, dd), 8.309 (1H, dd), 8.729 (1H, dd). IR (KBr) 1242 cm-1
(P═O) HRFAB MS for C22H34N4O2P, M.sup.+ 477.2419,
found 477.2400.

[0388]Small-scale solubility tests showed that this compound
(t-Bu-Dtp-OAt) was highly soluble in DMF and thus could be used in the
automated peptide synthesizer.

[0389]As indicated hereinabove, without wishing to be bound, it is
believed that during peptide coupling using uronium or phosphonium salts,
the N-protected carboxylic acid first reacts with the coupling reagent to
form an active ester, which then reacts with the amino component to give
the corresponding amide. Therefore, the speed of formation of such an
active ester is one of the important factors in evaluating the efficiency
of the coupling reagent. The model chosen hereinbelow involved conversion
of N-benzyloxycarbonyl-a-aminoisobutyric acid (CBZ-Aib-OH) to the
corresponding active ester in both DMF and CDCl3, as described in
the chemical equation hereinbelow.

##STR00076##

[0390]In the study, a comparison was made with derivatives of HOBt, HOAt
with HODhbt:

##STR00077##

[0391]The benzylic CH2 units of CBZ-Aib-OH (65.09) and active ester
(65.20) were monitored by NMR. Assignment of the peak at 65.20 in the
active esters was confirmed by authentic synthesis.

[0392]Because of the sterically hindered carboxyl group of Aib, activation
in the above equation is slow relative to the case of the proteinogenic
amino acids allowing different coupling reagents to be more closely
differentiated.

[0396](c) Active Ester Formation. To a solution of 0.1 mmol of CBZ-Aib-OH
and 0.1 mmol of the appropriate coupling reagent in 0.5 ml of CDCl3
or DMF, was added 0.1 mmol of DIEA. The mixture was immediately
transferred to an NMR tube which was placed in the probe of a Hitachi
R-1200 (60 MHz) NMR instrument. Integration of the 1H-NMR peaks at
δ 5.1 (acid) and 5.2 (active ester) as the reaction progressed at
the probe temperature (37° C.) allowed for rough determination of
the relative rates. The results are given in Table 1 and represent the
averages of at least two runs.

[0397]In the examples that follow, it is to be understood that the amino
acid sequence is presented in the amino to carboxy direction, from left
to right.

Example 20

[0398]To test for configuration control, three different peptides,
CBZ-Phe-Val-Pro-NH2, CBZ-Gly-Phe-Pro-NH2, and
CBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe (SEQ ID NO:1) were prepared and the loss
of configuration during the coupling was determined. An exemplary
procedure is given for a dipeptide as follows:

[0399]A. CBZ-Phg-Pro-NH2 As a standard protocol, 35.6 mg (0.125 mmol)
of CBZ-Phg-OH, 14.3 mg (0.125 mmol) of H-Pro-NH2, and 0.25 mmol of
base in 1 ml of DMF or other solvent was treated with 0.125 mmol of an
appropriate coupling reagent at 0° C. The mixture was stirred at
0° C. for 1 hour and at room temperature overnight. The resulting
mixture was diluted with 25 ml of EtOAc and washed with HCl (2×10
ml), 10% NaHCO3 solution (2×10 ml) and brine (2×10 ml),
and dried over MgSO4. An oily peptide residue was obtained after
removing solvent. The oily residue was redissolved in 1 ml of
CH2Cl2 and 20 ml of hexane was added. A white solid was
obtained after filtration. About 5 mg of this crude product, usually
containing both LL- and DL-forms of CBZ-Phg-Pro-NH2, was dissolved
in 4 ml of MeCN and directly analyzed by HPLC as described in Wenschuh,
et al., J. Org. Chem., 1995, 62, 405, the contents of which are
incorporated by reference.

[0400]B. CBZ-Phe-Val-Pro-NH2. The standard protocol as described
above for CBZ-Phg-Pro-NH2 was followed.

[0401]C. CBZ-Gly-Phe-Pro-NH2. The standard protocol as described
above for CBZ-Phg-Pro-NH2 was followed.

[0402]D. CBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe (SEQ ID NO 1.) As described for
CBZ-Phg-Pro-NH2, a solution of 45.6 mg (0.125 mmol) of
CBZ-Gly-Gly-Val-OH, 31.7 mg (0.125 mmol) of H-Ala-Gly-Gly-OMe.HCI and
49.6 μl (0.375 mmol) of TMP in 1 ml of DMF was treated with 0.125 mmol
of an appropriate coupling reagent at 0° C. The resulting mixture
was stirred at 0° C. for 1 hour and then at room temperature
overnight. The crude hexapeptide methyl ester was isolated by evaporation
of solvent followed by direct column chromatography using
MeOH/CHCl3/HOAc (3:7:0.1) as eluate. The crude material from the
column, containing both LL- and DL-forms of hexapeptide, was examined by
HPLC as described in Carpino, J. Org. Chem., 1994, 59, 695, the contents
of which are incorporated by reference.

[0404]As confirmed by the data herein, the new phosphorous-based OAt
derivatives of the present invention are much more effective in
preserving configuration than any of the other tested reagents, including
N-HATU. The best of the previously-described uronium/guanidinium salts
(N-HAPyU) sometimes equals the results of the new phosphorus esters, but
where differences are observed, the latter have proved more effective in
every case examined to date.

[0405]Among the results obtained from these data involving N-HAPyU, it was
found that for the new reagents DEPOAt and DPOPOAt, a one-equivalent
excess of proline serving as base gave the lowest epimerization levels
yet observed for the tripeptide CBZ-Phe-Val-Pro-NHZ in DMF (0.5%
LDL-isomer). Upon switching to other solvents, even greater differences
were found between the new phosphorus reagents of the present invention
and the related uroniunt/guanidinium salts. For example, in the special
structure-breaking combination solvent trifluoroethanol/trichloromethane
(TFE, TCM, 1:3), 12.2% of the LDL-form was obtained for DPOPOAt/TMP as
opposed to 38.5% for N-HATU/TMP. In CH2Cl2 in the presence of
TMP, 2.2% (DEPOAt) and 2.9% (DPOPOAt) were clearly better than values
observed for guanidinium reagents NHATU (9.3%) and N-HAPyU (5.3%).

[0406]In order to determine the coupling efficiency of diphenyl
phosphorochloridate (DPOPC1) various coupling conditions were used. It
was noted that without additive, DPOPC1 gave only a very small amount of
the desired peptide for both diisopropylethylamine and trimethylpyridine.
If one equivalent of HOAt (1-hydroxy-7-azabenzotriazole) was present, the
results were acceptable. Indeed the mixture DPOPC1/HOAt/Base, which
contains DPOPOAt as the active species, gave results which are comparable
to those obtained with the isolated reagent DPOPOAt.

[0407]The Tripeptide CBZ-Phe-Val-Pro-NH2 was also chosen as a model
to study loss of configuration associated with use of various reagents of
the present invention under solid phase conditions. In comparison with
results obtained in solution, the data show how much more difficult it is
to maintain configuration in the solid phase mode. The system involved
overnight coupling of four equivalents of CBZ-Phe-Val-OH onto
H-Pro-PAL-PEG-PS in the presence of 8 eqs. of trimethylpyridine in DMF,
cleavage of the tripeptide from the resin via trifluoroacetic
acid/H2O (9:1) over a period of 1 hour and separation of the
diastereomers as described for the solution system. Although extensive
loss of configuration occurs in all cases, the data show that the
effectiveness of the various coupling reagents follows the same order as
in solution, thus coupling reagent/LDL (%): DEPOAt/11.6, N-HAPyU/13.0,
N-HATU/13.6, DPOPODBt/19.4, DEPDBt/19.5, HDTU/24.2, N-HBTU/29.8.

Example 21

ACP Assembly via Stepwise Coupling on Solid Phase

[0408]In order to demonstrate the suitability of the
organophosphorus-based coupling reagents and compare their performance
with that of the corresponding uronium/guanidinium analogs in solid phase
syntheses, several syntheses of the ACP decapeptide segment 65-74
(H-Val-Gln-Ala-Ala-Asp-Tyr-Ile-Asn-Gly-NH2) (SEQ. ID. NO. 2) were
carried out.

[0409]The protocol is as follows: 150 mg of Fmoc-Gly-PAL-PEG-PS resin
(0.19 mmol/g, 0.0285 mmol) in a 10-ml disposable syringe fitted with a
Teflon filter was washed with CH2Cl2 (3×10 ml) and DMF
(3×10 ml) and deprotected with 20% piperidine in DMF (10 ml) for 7
min. The deprotected resin was washed with DMF (3×10 ml),
CH2Cl2 (3×10 ml) and again DMF (3×10 ml).
Preactivation was carried out for 7 min using 25.5 mg (0.04 mmol, 1.5
equiv) of Fmoc-Asn(Trt)-OH, 15.75 mg (0.04 mmol, 1.5 equiv) of DPOPOAt
and 14.89 μl (0.09 mmol, 3 equiv) of DIEA (diisopropylethylamine) in
0.15 ml of DMF in a 4-ml vial. Following the requisite preactivation
period (7 min), the solution of the activated amino acid was added to the
resin. The small vial was washed with 0.04 ml of DMF, and the washing was
also added to the above resin. The resulting resin mixture was allowed to
react at room temperature for 1.5 min. The loaded resin was washed with
DMF (3×10 ml) and the Fmoc group was deblocked with 10 ml of 20% of
piperidine in DMF for 7 min. Washing the deblocked resin with DMF
(3×10 ml), CH2Cl2 (3×10 ml) and DMF (3×10 ml)
was followed by an analogous coupling step with Fmoc-Ile-OH. Other amino
acids were coupled similarly and after the last coupling with Fmoc-Val-OH
and deblocking of the Fmoc group with 20% piperidine in DMF, the loaded
resin was washed with DMF (3×10 ml), CH2Cl2 (3×10
ml), EtOH (5 ml) and ether (5 ml). The resin was then treated with 10 ml
of 90% aqueous trifluoroacetic acid for 2 hours, filtered, and washed on
the filter with 10 ml of 10% trifluoroacetic acid in CH2Cl2 and
10 ml of CH2Cl2. The combined filtrates were evaporated to
dryness. The crude product was washed four times with anhydrous ether and
separated by centrifugation. The yield was calculated by the weight of
the crude product. For analysis 1 mg of the crude product was dissolved
in 1 ml of 0.1% aqueous trifluoroacetic acid and injected directly onto
the HPLC column for analysis. The procedure was repeated using the same
coupling agent until the peptide of SEQ ID NO. 2 was prepared.

[0410]This procedure was repeated using each of the coupling agents listed
in Table 3 for the preparation of the peptide of SEQ. ID. NO. 2. The
results are given in Table 3.

[0411]In this experiment, coupling times are shortened and excesses of
reagents are reduced in order to bring out differences among the various
reagents studied. Under these conditions, incomplete incorporations were
detected for Asn onto Gly, Ile onto Asn, Ile onto Asp, Val onto Gln and
Ala onto Ala or Asp. Analysis of the chromatograms indicated that the new
HOAt-based organophosphorus reagents are as effective as N-HATU under
these so-called "1.5×1.5" conditions with or without preactivation.
Under normal coupling conditions such as 4 eqs excess amino acid/30 min.
coupling time, all reagents worked well with the exception of HDTU.

Example 22

[0412]In the following example, reactions of the hindered active esters
CBZ-Aib-OXt with p-chloroaniline (PCA) were studied in CDCl3.
Approximate halftimes were determined by proton NMR analysis according to
the disappearance of the benzylic CH2 unit (δ 5.2) of the
active esters and appearance of the benzylic CH2 residue (δ
5.5) of the product.

[0413]CBZ-Aib-OXt Esters. The reaction of Z-Aib-ODhat with PCA is taken as
an example to demonstrate the standard method used in order to follow
aminolysis via an NMR protocol: To a solution of 47.9 mg (0.125 mmol) of
CBZAib-ODhat in 0.5 ml of CDCl3, was added 15.6 mg (0.125 mmol) of
p-chloroaniline (PCA). The mixture was immediately transferred to an NMR
tube, which was placed in the probe of a Hitachi R-1200 (60 MHz)
instrument. Integration of the 1H NMR peaks at δ 1.8 (CH3
residue of ester CBZ-Aib-ODhat) and 1.57 (CH3 residue of amide
CBZ-Aib-PCA) [or peaks at δ 5.2 (benzylic CH2 unit of ester
CBZ-Aib-ODhat) and 5.05 (benzylic CH2 unit of the product amide)] as
the reaction progressed at the NMR probe temperature (37° C.)
allowed for rough determination of the relative rates. The results given
in Table 4 are the average of at least two runs.

[0414]It was found that the ODhat ester is slightly more reactive even
than the OAt ester, which was previously found to be the most reactive
derivative among these esters. Interestingly, despite the structural
similarity between HODhat, i.e.,
3-hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazine, and HODhad, i.e.,
3-hydroxy-4-oxo-3,4-dihydroxy 5-azabenzo-1,3-diazine the reactivities of
the corresponding active esters are very different. Without wishing to be
bound, it is believed this may be due to the presence or absence of
additional neighboring group effects promoted by the presence or absence
of a nitrogen atom substituted at the 2-position. On the other hand,
comparison of the OBt and ODhad esters demonstrates the importance of the
neighboring carbonyl group.

Example 23

[0415]In this example, a second model was used. Pivaloyl esters in the
presence of various comparative coupling agents were treated with
benzylamine and N-methylbenzylamine, which led to the formation of amides
of the following formula:

##STR00078##

[0416]Approximate halftimes for these reactions were determined by proton
NMR analysis, according to the disappearance of the methyl peak (δ
1.5) for pivaloyl-OXt and the appearance of the methyl peak for products
XXVa(δ 1.2) or XXVb(δ 1.3). The protocol is as
follows:

[0417]Me3CCOOXt Esters. As in the case with Example 22, the reaction
of pivaloyl ester with N-methyl benzylamine is taken here as an example
to demonstrate the methodology used: To a solution of 31.0 mg (0.125
mmol) of Me3CCOODhat in 0.5 ml of CDCl3, was added 15.1 mg
(16.1 nl, 0.125 mmol) of PhCH2NHMe. The mixture was immediately
transferred to an NMR tube, which was placed in the probe of a Hitachi
R-1200 (60 MHz) instrument. Integration of the 1H NMR peaks at
δ 1.5 (CH3 residue of ester Me3COODhat) and 1.3 (CH3
residue of amide) as the reaction progressed at the NMR probe temperature
(˜37° C.) allowed for rough determination of the relative
rates. The results given in Table 5 are the average of at least two runs.

[0419]In the case of benzylamine all reactions were rapid whereas in the
case of the more hindered N-methyl derivative, clear reactivity
differences were seen according to the following order:
ODhat>OAt>ODhbt>ODhad>OBt. Again the greater reactivity of
the HODhat ester relative to that derived from HOAt is seen.

Example 24

[0420]Another comparative study was run to compare relative rates of
coupling processes involving the reaction of CBZ-Aib-OH with
p-chloroaniline (PCA) in the presence of a coupling reagent. Because
formation of intermediate Z-Aib-OXt is usually very fast, halftimes are
determined by disappearance of the benzylic CH2 residue (δ
5.2) of the active ester and appearance of the benzylic CH2 unit
(δ 5.05) of product CBZ-Aib-PCA unless otherwise noted. The
protocol for the preparation of this product is the same as described in
Example 23. Approximate halftimes are collected in Table 6. In this case
various solvent systems were examined

[0421]Interestingly in all solvent systems examined except for DMF, the
new coupling reagent was found to be more reactive than N-HATU. In
CDCl3, HDATU is at least six times as reactive as N-HATU and about
eight times as reactive as HDTU. So far, in every case tested HDATU was
shown to be significantly more reactive than HDTU.

Example 25

[0422]In order to test the configuration-retention effectiveness of the
additives HODhat and HODhad, and the coupling reagents HDATU, HDADU,
HDAPyU, HDPyU, PyDAOP, and PyDOP, the following model peptide systems
were examined These involve a [1+1] stepwise coupling, and three [2+1]
and one [3+3] segment couplings.

[0423]Test couplings were carried out as described previously in Example
22 and in L. A. Carpino, et al., J. Org. Chem. 1990, 61,2463, for
CBZ-Phg-ProNH2, CBZ-Phe-Val-Pro-NH2, CBZ-Gly-Phe-Pro-NH2
and CBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe substituting the coupling reagent
listed hereinbelow in the tables and the protocols therein, the results
of which are incorporated by reference. For Boc-Gly-Leu-Phe-OBzI,
(Bzl=benzyl) 60.6 mg (0.21 mmol) of Boc-Gly-Leu-OH, 85.45 mg (0.20 mmol)
of H-Phe-OBzl.pTsOH and 0.22 mmol of an appropriate coupling additive
(HOXt) were dissolved in 1 ml of DMF or trifluoroethanol/trichloromethane
(1:3 v/v). To the mixture, a solution of 34.2 mg (0.22 mmol) of EDC
(1-ethyl-3-3'-(dimethylamino)-propyl)carbodiimide in 1 ml of DMF or
trifluoroethanol/trichloromethane was added and the whole mixture was
stirred at room temperature overnight. The resulting mixture was diluted
with 25 ml of EtOAc and washed with 1 N HCl (2×10 ml), 10%
NaHCO3 (2×10 ml) and brine (2×10 ml), and dried over
MgSO4. After removal of solvent, the solid was weighed to determine
the yield. The solid was then stirred with 2 ml of 50% trifluoroacetic
acid in a methylene chloride solution for 2 hours to deblock the
BOC-group. The trifluoroacetic acid and CH2Cl2 were then
removed in vacuo and 20 ml of anhydrous ether was added to the oily
residue, and the mixture was stored at room temperature overnight. The
white precipitate which had separated was collected by filtration and
washed with ether. About 5 mg of the crude product, containing both LL-
and DL-forms of XXX was dissolved in 4 ml of MeCN and directly analyzed
by HPLC using a reversed-phase Waters C-18 column, with elution by a
linear gradient over 20 min of 0.1% trifluoroacetic acid in MeCN and 0.1%
aqueous TFA from 1:9 to 11:9, at a flow rate of 1.0 ml/min. The retention
times for the LL and DL-forms of XXX were 17.3 and 17.9 min,
respectively. The results are as follows:

[0424]For the sensitive coupling of the urethane-protected CBZ-Phg-OH to
H-Pro-NH2 to give XXVI, HDATU was more effective in preserving
configuration than HDTU and N-HBTU, but not better than N-HATU. Curiously
with this system, use of the base diisopropylethyl amine (DIEA) proved
more satisfactory than collidine (TMP), a result that is rarely observed
in the case of the corresponding segment couplings. Results are collected
in Table 7.

[0425]With carbodiimide reagents in the solvent
trifluoroethanol/trichloromethane, HODhat was even more effective than
HODhbt. Thus, EDC/HODhat gave 0.5% of the DL-isomer, whereas EDC/HODhbt
led to 1.3% of the same form. For DCC/HODhat and DCC/HODhbt in the
presence of 1 equivalent of trimethylpyridine, the figures were 0.4% and
0.8%, respectively.

[0426]For the well-studied segment coupling of CBZ-Phe-Val-OH to
H-Pro-NH2 leading to tripeptide XXVII, the results are tabulated in
Table 8.

[0428]For the preparation of tripeptide XXVIII, HDATU was similar to or
even slightly more effective than N-HATU.

[0429]With respect to the test tripeptide XXX, the coupling of H-Phe-OBz1
TosOH with Boc-Gly-Leu-OH in the presence of EDC/additive (coupling
agent) in various solvents gave a product Boc-Gly-Leu-Phe-OBz1 which was
BOC-deblocked via 50% TFA/CH2Cl2 to give the crude tripeptide,
which was directly analyzed by HPLC.

[0430]In the EDC-mediated synthesis of XXX carried out in
trifluoroethanol/chloroform (1:3 v/v), the three additives HODhat,
HODhbt, HOAt were found equally effective with less than 0.1%
epimerization being observed. Upon switching to DMF as solvent,
differences, although small, could be noted. Results are shown in Table
10.

[0431]Following preliminary studies with simple di- and tripeptide models,
XXVI, XXVII, XXVIII and XXX, a test peptide XXIX was assembled. The
coupling of Z-Gly-Gly-Val-OH to H-Ala-Gly-Gly-OMe is a sensitive test for
the nature of both coupling reagent and base. Results for the reaction in
DMF, in the presence of collidine are gathered in Table 11. HDATU was
found to be more effective in preventing loss of configuration at valine
than N-HATU and other coupling reagents. Epimerization levels up to 8.2%
of the DL-form were noted according to the order:
HDATU<N-HATU<HDTU<N-HBTU.

[0432]In order to demonstrate the suitability of the new HODhat-based
coupling reagent HDATU and compare its performance with that of the
corresponding guanidinium/uronium analogs N-HATU and HDTU in solid-phase
syntheses, 30 syntheses of the ACP segment
H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-GlyNH2, (SEQ. ID. No. 2) were
carried out by an Fmoc/tert-butyl protection scheme as described in
Example 22. Polyethylene glycol-polystyrene (PEGPS)-resin bearing
Fmoc-glycine was used as solid support. Peptide elongation was performed
manually, coupling times being shortened and excesses of reagents being
reduced in order to bring out the differences among the various coupling
reagents studied. Under these conditions, incomplete incorporations were
detected for Asn onto Gly, Ile onto Asn, Ile onto Asp, and Val onto GIn.
Peptide purity was judged by reverse-phase HPLC analysis, after cleavage
from the resin with TFA-H2O (9:1) for 2 hours at room temperature.
The results are collected in Table 12.

[0433]Analysis of the chromatograms indicated that HDATU is far more
effective than HDTU under all conditions examined, and more effective
even than N-HATU in many instances. Methylene chloride was found to be a
particularly suitable solvent for HDATU-mediated ACP synthesis. Thus,
under so called "1.5×1.5" conditions in CH2Cl2, HDATU
gave the decapeptide in a purity of 47%, whereas N-HATU and HDTU led to
only 21% and 4% of the desired product, respectively. When a 4-equiv
excess of reagents and a 3-min coupling time were used, 86% of acyl
carrier protein (ACP) was obtained for HDATU, compared with 78% and 31%
for N-HATU and HDTU, respectively.

[0434]In DMF under "1.5×1.5" conditions, the performance of HDATU
may not have been as efficient as N-HATU with or without preactivation.
In addition, for preparation of model pentapeptide
H-Tyr-Aib-Aib-Phe-Leu-NH2 (SEQ ID NO:3) which incorporates the
highly hindered Aib-Aib unit, whether in DCM or DMF, HDATU was not able
to equal the results obtained with N-HATU. For example, with 4
equivalents excess acid, 7 min preactivation and 30 min coupling time
HDATU gave in DMF a peptide of 31% purity, whereas with NHATU the purity
was 91%.

[0435]However, in general HDATU was found to be the better reagent under
normal conditions. Thus, while using a 2-equiv excess of reagents without
preactivation for a 5-min coupling, ACP was obtained in 97% purity by
HDATU, whereas the corresponding values were 94 and 81% for N-HATU and
HDTU. With 4-equiv/30 min coupling conditions with a 7-min preactivation
time, excellent purity (95%) was obtained for HDATU, whereas with N-HATU
and HDTU, the ACP purity was only 86 and 62%, respectively.

[0436]When DIC/HODhat was used as a coupling reagent, satisfactory results
were also obtained. Although not suitable under stringent conditions
("1.5×1.5"), HODhat could be used as an excellent catalyst and
indicator in Fmoc-amino acid pentafluorophenyl (Pfp) ester couplings
under normal conditions. A bright-yellow-to-orange-red color change was
noted which is much clearer than the color change from bright-yellow to
pale-yellow observed with HODhbt. In DMF under conditions involving 3
equiv of pentafluorophenyl-ester and a 30-mm coupling time, both HODhat
and HODhbt gave the desired ACP product in a purity of over 85%.

[0438]Method B. Under an atmosphere of dry N2, 0.1854 g (0.5 mmol) of
Fmoc-Ile-Cl was added with stirring to a solution of HODhat (0.0821 g,
0.5 mmol) and DIEA (95.8 μl, 0.55 mmol) in 10 ml of CH2Cl2
at 0° C. Stirring was continued at 0° C. for 30 min and
then at room temperature for 5 hours. The resulting light yellow solution
was diluted to 30 ml with CH2Cl2 and washed quickly with
ice-cold brine (2×15 ml). After drying over MgSO4 and removing
the solvent, the light yellow sticky solid was recrystallized twice from
CH2Cl2-ether-hexane to give the analytically pure
above-identified ester as a white solid: mp 161-162° C.; NMR and
IR spectra were identical with those of the sample obtained by Method A.

Example 28

[0439]Utilizing the procedure described in footnote a of Table 2 of the
article by Carpino, et al., J. Org. Chem. 1995, 60, 3561, the contents of
which are incorporated by reference, the coupling of CBZ-Phe-Val-OH with
H Pro-NH2 to form CBZ-Phe-Val-Pro-NH2 was investigated using
various coupling reagents. Some of the coupling reagents used were those
described elsewhere in the art while others used were coupling agents of
the present invention. More specifically, for carbodiimide couplings,
0.105 mmol of Z-Phe-Val-OH, 0.1 mmol of H-Pro-NH2, and 0.11 mmol of
the coupling reagent noted hereinbelow in the table were dissolved in 1
ml of DMF or 1.3 ml of TFE/TCM (trifluoro ethanol/chloroform) (1:3), and
the solution was cooled in an ice bath and treated with 0.11 mmol of EDC,
EDC-HC 1, or DCC. If a base is added, the number of equivalents is given.
The mixture was stirred at 0° C. for 1 hour and at room
temperature overnight. The mixture was diluted with 25 ml of EtOAc and
extracted with 1 N NCl (2×5 ml), 1 N NaHCO3 (2×5 ml),
and saturated NaCl (2×5 ml), dried with MgSO4, the solvent was
removed, and the crude peptide was directly analyzed by HPLC. For onium
salt couplings, 0.125 mmol of the acid, 0.125 mmol of amide, and 0.25
mmol of base in 1 ml of DMF was treated with 0.125 mmol of coupling
reagent at 0° C. and the reaction mixture was stirred at 0°
C. for 1 hour and at room temperature for 2-3 hours, after which the
workup followed that described herein. In cases where an additive is
used, one or more equivalents of base (given in parentheses) may be
added. The amount of loss of configuration, as indicated by the presence
of LDL epimer was determined. The results are tabulated in Table 13.

[0440]Using the procedure as described in the article by Camino, et al. in
Tetrahedron 1999, 55, 6813, the contents of which are incorporated by
reference, the coupling of Fmoc-Asp (t-Bu)-Phe-OH and F-moc-Lys
(BOC)--PAL-PEG to form Fmoc-Asp-(t-Bu)-Phe-Lys(-Boc)-PAL-PEG was
conducted. Some of the coupling reagents used were those described
elsewhere while others used were those of the present invention. Coupling
reactions were carried out by deblocking 50 mg of H-Lys(Boc)-PAL-PEG-PS
resin by means of 20% piperidine/DMF for 7 min, washing the resin with
DMF, DCM and DMF (3×5 ml each) and then adding a 5-fold excess
(0.0475 mmol) of Fmoc-Asp(O-t-Bu)-Phe-OH (26.5 mg), a 5-fold excess of
the coupling reagent noted in Table 14 and 11.5 mg (0.095 mmol) of TMP or
12.3 mg of DIEA (10-fold excess) of the base, if any. In each case the
coupling reagent and the base were dissolved in 0.2 ml of the solvent and
the resulting solution added to the resin in a small syringe which served
as the reactor. Dissolution required about 1 min or less and care was
taken to add the solution as soon as possible after everything dissolved.
This method is referred to as the "low preactivation" method. Where
preactivation was involved, the times are recorded. The mixture was
stirred gently every 10 min with a Teflon rod for approximately 1 hour
and then allowed to stand for 12 hours after which the resin was washed
with DMF and DCM (3×5 ml each) and deblocked by treatment with 3 ml
of TFA/H2O (9:1) for 11/2 hour at room temperature. The solvent was
removed in vacuo and the residue dissolved in CH3--CN for direct
injection onto an HPLC column under the following conditions: 4μ 60 A,
C18 Waters Nova-pak column, 3.9×150 mm; flow rate 1 ml/min;
Waters 996 PDA detector; linear gradient 10/30 in 20 min and then
isocratic 30/70 for 20 min with CH3CN/H2O/0.1% TFA; Rt
(LLL-) 28.5 min, (LDL-) 30.5 min. The amount of loss of configuration as
indicated by the presence of LDL epimer was determined The results are
tabulated in Table 14.

[0441]Utilizing the procedure of Example 29, and utilizing
CBZ-Gly-Gly-Val-OH and H-Ala-Gly-Gly-PAL-Peg,
CBZ-Gly-Gly-Val-Ala-Gly-Gly-PAL-PEG (Sequence ID 4) was formed using
t-Bu-DtP-OAt of the present invention and O-HATU. The amount of loss of
configuration was determined by measuring the amount of LDL epimer
formed. The results are indicated in Table 15 hereinbelow.

[0442]The high coupling efficiency of the coupling reagents of the present
invention including t-Bu-Dtp-OAt was emphasized by solid phase synthesis
of ACP decapeptide under the so-called "1.5×1.5" protocol. Under
these demanding conditions the coupling efficiency of various coupling
reagents can be easily brought out. The couplings are carried out for 1.5
minutes using a 1.5-eq excess of protected amino acids and 1.5 eq of
coupling reagent in the presence of 3 eq of base.

[0443]For manual solid phase syntheses of ACP under the 1.5×1.5
protocol, using the procedure of Carpino, et al., in J. Chem. Soc. Chem.
Comm, 1994, 201, the contents of which are incorporated by reference and
using O-HATU, Dtp-OAt and t-Bu-Dtp-OAt the purity of the crude peptide
was 76%, 60% and 74%, respectively. The new phosphorus-based coupling
reagent therefore at least equals the effectiveness of O-HATU, considered
the best of the previously described reagents.

[0444]The above preferred embodiments and examples are given to illustrate
the scope and spirit of the present invention. These embodiments and
examples will make apparent to those skilled in the art other embodiments
and examples. These other embodiments are within the contemplation of the
present invention. Therefore, the present invention should be limited
only by the appended claims.

Patent applications by Chongwu Zhang, Dayton, NJ US

Patent applications by Jusong Xia, Moore, SC US

Patent applications by Louis A. Carpino, Amherst, MA US

Patent applications in class Four or more ring hetero atoms in the polycyclo ring system

Patent applications in all subclasses Four or more ring hetero atoms in the polycyclo ring system